安颤灵对帕金森病模型内质网应激与泛素—蛋白酶体系统的影响
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摘要
目的
帕金森病(Parkinson disease,PD)是渐行性神经退行性疾病,表现为黑质纹状体中多巴胺能神经元的进行性死亡,神经元胞浆内包涵体Lewy小体增多。PD的病因至今仍未完全明确,但公认的机制有兴奋性毒性和能量代谢紊乱、氧化应激、线粒体活性下降等功能紊乱。环境因素比如杀虫剂和其他因素也可以诱发散发性PD,而散发性PD占全部病例了90%以上。最近研究表明,PD疾病的发生与内质网应激和泛素-蛋白酶体系统有关。本课题结合国际帕金森病研究的最新进展和动态,采用腹腔注射MPTP诱导模拟人的PD慢性病程的动物模型,研究内质网应激与泛素-蛋白酶体系统与帕金森病发病的关系；同时进行天然中药新药安颤灵的药效学作用机理研究,为其进一步中药新药开发提供科学依据。
方法
1.动物实验：
(1)建立PD模型及实验分组：
选取健康雄性c57BL/5J小鼠60只(9周龄),随机分成5组,分别为PD模型组12只,予小鼠腹腔体内注射MPTP(30mg/kg.d),同时蒸溜水灌胃与安颤灵组等体积。正常对照组12只,蒸溜水灌胃与模型组相同,腹腔注射改为相同体积的生理盐水(30mg/kg.d)。安颤灵组(分高、中、低剂量组),每组各12只,该组在模型组基础上将灌胃的蒸溜水改为以上不同剂量的中药煎液。
(2)动物行为学观察：
注射后7d、14d和21d按0.25mg/kg腹腔注射阿扑吗啡,观察旋转行为的变化,共记录30mi n取平均值。同时注意观察小鼠震颤、活动迟缓、抓握、嗅探等异常行为。
(3)黑质区TH、GFAP.OX-42免疫组化检测：
注射后第21天,在2.5%戊巴比妥钠麻醉下,从左心室插管灌注4%多聚甲醛,之后断头取脑,经系列生化固定后。自近黑质部位起连续冠状切片,挑取对称的黑质区切片。行TH、GFAP、OX-42免疫组织化学染色,观察中脑黑质酪氨酸羟化酶(TH)阳性细胞,星形胶质细胞(GFAP)及小胶质细胞(OX-42)的变化,以了解DA能神经元损毁情况以及是否选择性地损毁多巴胺能神经元以及小胶质细胞的激活。切片再经系列生化免疫标记,置显微镜观察,计数阳性细胞数。
(4)a-synuelein、Parkin和泛素(ubiquitin)的免疫组化检测：
通过免疫组织化学染色法观察DA能神经元lewy小体的主要成分a-共核蛋白(a-synuelein)、Parkin和泛素(ubiquitin)的表达。切片经系列生化免疫标记后,显微镜观察,显微图象分析检测。模型组、正常组、安颤灵组处理时间和剂量完全相同。
(5)RT-PCR检测黑质区组织Caspase-12mRNA的表达：
GenBank检索小鼠Caspase-12序列,自行设计引物,由上海Sangon公司合成。Caspase-12引物(506bp)上游：5'-AGGCAACTCTCATGCAGTTC-3'(1319-1338bp),下游：5'-AACCATGTATGCCA GAG AC C-3'(1847-1866bp)。取总RNA4ul,加入Olig (dT)1ul,65℃15min,立即置冰上；加入10XRT缓冲液2μl,dNTP2ul,25mmol/L MgC122μl,RNA酶抑制剂1μl,A-MV1μl,加DEPC处理水至总体积20μl,42℃60min逆转录；取逆转录产物cDNA4μl,加入10%PCR缓冲液5μl,dNTP1μl, Caspase-12上游、下游引物各2μl(20pmol),Taq酶(5U/μl)0.25μ l,25mmol/LMgC123μl,加无菌双蒸水至总体积50μl。PCR条件：94℃5min,94℃40s,55℃40s,72℃90s,35个循环；72℃10min。取PCR扩增产物10μl行琼脂糖凝胶电泳,经凝胶成像系统扫描Caspase-12灰度值.。比较各组黑质区组织Caspase-12mRNA的表达。
(6)Western blot检测黑质区组织Caspase-12, CHOP、Bip/GRP78蛋白的表达：
50mg小鼠黑质组织用1%TritonX-100加蛋白酶抑制剂裂解,用牛血清白蛋白方法检测蛋白含量。每一样品取总蛋白30ug上样,用12%不连续聚丙烯酞胺凝胶电泳,转蛋白至硝酸纤维素膜；5%脱脂奶粉封闭2h；加进入Caspase-12、CHOP、Bip/GRP78第一抗体轻摇2h后4℃过夜；Tris磷酸盐缓冲液洗膜15min,共3次；加人辣根过氧化物酶标记的第二抗体37℃1h,Tris磷酸盐缓冲液洗膜15min,共3次。化学发光试剂作用后X胶片曝光,经显影、定影等处理后观察结果。用凝胶成像系统扫描图像,对其灰度分析,比较各组黑质区组织Caspase-12, Bip/GRP78蛋白的表达。
(7)数据统计：数据统计采用均值(x)±标准差(s),采用SAS软件包行单因素方差分析检验,以P<0.05表示有显著性差异。
2.离体实验：安颤灵对H2O2诱导PC12细胞凋亡及内质网应激的影响
(1)细胞株及培养体系：
Pc12细胞是小鼠肾上腺嗜铬细胞瘤细胞株,由中国科学院上海细胞生物学研究所提供。细胞接种在预先铺过鼠尾胶的培养皿中,用含有10%胎牛血清和10%马血清的DMEM培养基(美国Gibco BRL公司),同时加入2mmol/L谷氨酰胺、100u/ml链霉素和100u/ml青霉素,在温度37℃、5%CO2、饱和湿度的cO2培养箱中培养。隔天传代细胞1次,实验所用的细胞均处于对数生长期,且存活细胞百分率均在95%以上(台盼兰拒染法)。
(2)实验分组和处理：
在24孔培养板上,按每孔1．25×105个细胞的浓度加入Pc12,在DMEM中培养4h后,加入高、中、底不同浓度的安颤灵含药血清(50,100,200ug/ml)预处理3d,然后将细胞暴露与200mMH2O2中。经12h共同培养后,测定细胞的凋亡／死亡和存活情况。设正常和模型对照,实验重复3次。
(3)PC12细胞DNA倍体的分析：
在100μl样本液中加人含50mg/L碘化丙啶(美国BD公司)和50mg/L核糖核酸酶A(上海生工生物工程公司)的染液500μl,避光37℃孵育30min,流式细胞仪采集数据进行亚二倍体峰(subGl)的百分比分析。每份标本利用CellQuestTM软件分析10000个细胞,结果用百分比表示。
(4)凋亡细胞染色和荧光观察：
弃去介质和处理因素,用1×D-PBS轻轻清洗细胞一次。直接在细胞上添加适当量(约在24孔培养板上按50uL/孔,在12孔培养板上按100uL/孔或6孔培养板上按150uL/孔)的联合染液(2mM Calcein AM和4mM Ethidium Homodimer-1),在室温下孵育30分钟。标记的存活和死亡细胞用荧光显微系统(Zeiss Axiovert135M, Mercury Lamp, Carl Zeis HBO100W/Z),以485nm和590nm立即观察和拍照。
结果
1.动物实验结果：
1.1造模及行为学结果：腹腔注射MPTP后,PD模型组小鼠于第1次注药后10-20min均出现不同程度的震颤、竖毛、翘尾,上述症状持续4-5h后逐渐减轻,但仍有活动减少,动作迟缓等变化。治疗前模型组和安颤灵组组间的旋转圈数无显著性差异(P>0.05)。治疗后,实验第7天、第14天和第21天安颤灵组小鼠旋转次数逐渐减少,分别与模型组相比,差异显著(P<0.05、P<0.01、P<0.01)。悬挂行为结果显示：实验前1天(即首次注射MPTP前1天)观察小鼠悬挂行为,模型组、安颤灵组小鼠评分分别与对照组相比,无差异(P>0.05、P>0.05)；实验第7天、第14天和第21天观察模型组小鼠悬挂行为,其评分明显降低,分别与对照组相比,差异显著(P<0.01、P<0.05)；实验第14天和第21天安颤灵组小鼠悬挂实验评分明显升高,分别与模型组相比,差异显著(P<0.01、P<0.01)。实验前1天观察小鼠爬杆行为,模型组、安颤灵组小鼠评分分别与对照组相比,无差异(P>0.05、P>0.05)。注射MPTP第7天、第14天和第21天观察模型组小鼠爬杆行为,其评分明显降低,分别与对照组相比,差异显著(P<0.01、P<0.05、),提示造模成功。实验第14天和第21天安颤灵组评分明显升高,分别与模型组相比,差异显著(P<0.05、P<0.05)。
1.2.免疫组化、RT-PCR、Westernblot检测结果：
1.2.1免疫组化结果：PD模型组小鼠黑质区TH表达显著降低(P<0.01),GFAP、OX-42表达增多。经安颤灵灌胃处理的各组小鼠TH较模型组增多(P<0.01),而GFAP、OX-42阳性细胞数无明显变化。与模型组相比,中药安颤灵组a-synuclein.Parkin、ubiquitin阳性细胞数均减少(P<0.01)。
1.2.2RT-PCR结果提示,腹腔注射MPTP可诱导小鼠脑黑质区神经元的内质网应激反应,而安颤灵对这一应激反应有一定的缓解作用。
1.2.3.Western blot检测结果显示：模型组小鼠黑质区Caspase-12、CHOP蛋白表达水平较正常对照组显著上调(P<0.01),提示黑质区神经元的内质网应激反应提高。安颤灵组Caspase-12、CHOP蛋白表达水平较模型组明显降低(P<0.05)。模型组小鼠黑质区组织GRP78/Bip蛋白的表达较正常对照组上调,安颤灵组GRP78/Bip蛋白的表达较模型组进一步上调(P<0.05)。
2.离体实验结果：
实验光镜下观察发现,对照组PC12细胞排列整齐,贴壁生长旺盛,形态规则,呈长梭形或长棱形,有长短不等的类似神经轴突的伪足,交织成网状。H2O2损伤后的模型组细胞,排列紊乱,贴壁不牢,伪足缩短火消失,大量细胞悬浮,碎裂,死亡。安颤灵含药血清培养细胞变化较模型组明显减弱。细胞排列尚整齐,悬浮细胞较少,可见明显伪足。与大、小剂量组相比,中剂量组细胞的存活率显著升高(P<0.01)；与正常组相比,模型组细胞凋亡指数明显增加(P<0.01),a-synuclein、阳性细胞数亦均增多,而TH阳性细胞明显减少；与H2O2组相比,安颤灵组细胞凋亡指数明显降低,a-synuclein阳性细胞数均减少,TH阳性细胞明显增加(P<0.01)。H2O2模型组PC12细胞与正常对照组相比细胞周期明显左移。在细胞G1/Go期前有明显的亚二倍体峰(subGl-Go),处于此期的细胞比率为(94.50±21.3)%,而正常对照组处于此期的细胞比率为(1.36±2.0)%。而且H2O2模型组G1/G0期及所占的比率明显低于正常对照组,G2期及M期的细胞所占的比例也减少,在细胞周期图中代表这两期的峰几乎不可见。而同时给予不同浓度安颤灵含药血清(2.5%、5%、10%)孵育后,Pc12细胞subG1-Go百分比分别为(82.90±12.6)%、(69.34±15.7)%、(51.63±6.8)%。
结论
中药安颤灵小鼠行为学表明,采用MPTP注射法,造模成功；安颤灵能提高肢体运动协调能力,对MPTP诱导所致的PD模型小鼠PD运动体征具有明显的改善作用。ESR和UPS可能是PD多巴胺能神经元凋亡的关键因素,内质网应激的早期调节过程就存有泛素-蛋白酶体系统对异常蛋白的降解活动。ERS和UPS有相互促进作用,以达到内质网蛋白合成与降解的动态平衡,保证内环境的稳定。中药安颤灵就是通过黑质区组织Caspase-12mRNA的表达和Caspase-12, CHOP、Bip-GRP78蛋白的表达靶点的干预来实现对内质网应激反应(ESR)影响和改善,减少细胞毒性蛋白的合成。通过a-synuelein.和泛素(ubiquitin)蛋白的表达靶点的干预来实现对UPS的影响和改善,提高发现和破坏这些装配和折叠错误的蛋白的能力,使它们及时降解,消除细胞毒性蛋白,达到改善保护黑质区DA能神经元神经细胞,减少细胞凋亡。同时,对H202诱导的神经元ERS与凋亡反应具有显著地拮抗作用,从而达到保护细胞的作用。提示,H2O2能诱导细胞凋亡,而安颤灵可抑制这一细胞凋亡过程。腹腔注射MPTP可诱导c57小鼠黑质区神经元的内ESR,而安颤灵对这一应激反应具有明显拮抗作用,且该作用在一定浓度范围内呈现剂量依赖性。安颤灵可抑制PD小鼠模型黑质细胞的凋亡,并可改善UPS的功能。表明细胞内质网生成的毒性蛋白质a-synuclein.减少,毒性蛋白质的泛素降解通路正常或提高。表明ERS功能受到保护干预和UPS的功能活性良好。提示安颤灵可抑制PD小鼠模型黑质细胞的凋亡,可保护和改善ERS及UPS的功能。
Objectives
Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of the dopamine (DA) neurons in the substantia nigra pars compacta; accumulation of eosinophilic intraneural inclusions called Lewy bodies in the brain. Etiology of cell death still remains unknown, but it may result from generation of excitotoxicity, metabolic disorder, free radicals, oxidative stress and mitochondrial abnormality; also, environmental factors, such as exposure to the pesticides, has long been suggested as a risk factor for sporadic PD of which account for up to90%of all cases of PD. Ongoing researches suggest that the cause of PD was associated with the endoplasmic reticulum stress (ERS) and Ubiquitin-Protea some System (UPS). Based on these findings, together with the latest international studies, this present study aimed to investigate the relationship between ERS-UPS and etiology of PD and to elucidate an effect of Anchanling (a Chinese medicine, used as clinical therapy for early stage PD) on the ERS-UPS in PD mice model through establishing a mouse PD model induced by intraperitoneal injection of MPTP; furthermore the study was to explore the pharmacodynamic mechanism of Anchanling and to provide the development of new Chinese medicine for a treatment for PD with more scientific evidence.
Materials and Methods
1. The animal experiment:
(1) PD model establishment and experimental groups:
A total of60healthy male C57BL/5J mice (9-week-old) were selected and then was randomly divided into5groups:the PD model group (12), control group (12) and ACL group (high-dose, medium-dose and low-dose; every group contained12mice). At the start of experiment, the mice from PD model group were intraperitoneally injected with MPTP (30mg/kg. d) and were administered intragastrically perfusion with distilled water; The control group was intraperitoneally injected with normal saline (30mg/kg. d) and intragastrical method was the same as in the PD model group; three sub-group of ACL group were injected with the same volume of MPTP as did in the PD model group and then administrated intragastrically with three different volume of ALC decoction (high-dose, medium-dose and low-dose, respectively).
(2) Animal behaviour observation:
All the mice were intraperitoneally injected with0.25mg/kg apomorphine every seven days from the beginning of experiment postoperatively and induced rotational behaviour were observed for30minutes after injection and the rotations were recorded for three weeks (7d,14d and21d). Meanwhile, other abnormal behaviours, such as tremor, bradykinesia, grasping and sniffing were observed as well.
(3) Immunohistochemistry test on Substantia nigra tyrosin hytroxylase (TH), GFAP and OX-42:
Three weeks after the administration, the mice were anesthetized by intraperitoneal injection of2.5%pentobarbital, followed by perfusion of4%papraformaldehyde via left ventricular incubation. Brains were harvested and positioned in a stereotaxic apparatus. Serial coronal sections were prepared originating from the substantia nigra using freezing microtome and the symmetrical sections from the substantia nigra were selected for subsequent assay. Immunohistochemistry staining on TH, GFAP and OX-42was applied to observe the morphological change occurring among TH-positive cells, GFAP and OX-42so as to investigate damage level of dopaminergic (DA) neurons and to recognise whether GFAP selectively attack DA neurons as well as to identify whether OX-42has been activated. The sections were reused for immunohistochemical labeling and the numbers of TH-positive cells were counted under the fluorescent microscope.
(4) Immunohistochemistry test on a-synuclein, Parkin and ubiquitin:
a-synuclein, Parkin, and ubiquin, as the main components of Lewy bodies, their protein expression could be observed via immunohistochemical staining. After a serial of immunohistochemical processes (such as rinse, blocking and makers) sections were placed under the fluorescent microscope to count the above three proteins positive cells and then assay via microimage analysis. All the procedures including the doses of reagents and time consuming applying in the PD model group, control group and ACL group were the same.
(5) RT-PCR assaying the expression of substantia nigra tissue Caspase-12mRNA
The nucleotide sequences were based on the published Caspase-12(retrieved GenBank) and the self-designed primers was synthesized by Shanghai Sangon Company. Caspase-12primer (506bp) displayed as following, forward:5'AGGC AACTCTCATGCAGTTC-3'(1319-1338bp), reverse:5'-AACCATGTATGCCAGAG AC C-3'(1847-1866bp). First-strand complementary DNA was synthesised as following:taking4ul total RNA and adding withlu101ig (dT) at65℃for15min, then immediately placing on the ice; adding2μl10xRT buffer solution,2ul dNTP,2μl125mmol/LMgCl,1μ L ribonulclease inhibitors,1μl L A-MV, and adding DEPC-treated water to make total volume reach20μl and then keeping60min at42℃for reverse transcription; The PCR mixture contained4μl cDNA,5μl10%PCR buffer solution,1μ1dNTP,2μl (20pmol) forward primers of Caspase-12and2μl (20pmol) reverse primers of Caspase-12,0.25UlTaq DNA polymerase (5U/μl),3μl125mmol/L MgC12, and sterile double-distilled water in a final volume50μl was also prepared. The procedure of PCR was performed at94℃for5min, followed by35cycles at94℃for40s,55℃for40s,72℃for90s and extension at72℃for10min. Next,10μl PCR products were electrophoresed on agarose gel and the gels image were examined and analysed by gel documentary system to detect Caspase-12grey value. Finally, to compare the Caspase-12mRNA expressions among PD model group, control group and ACL group.
(6) Western Blotting analysis of Caspase-12, CHOP, Bip/GRP78
50mg mice substantia nigra tissue was employed with1%TritonX-100and protease inhibitors to encourage cleaves of the cells, with bovine serum albumin (BSA) to detect protein concentrations of cells lysates. An equal amount of protein for each sample (30ug in total) were separated by using12%sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with5%skimmed milk powder for2h. After blocking, the primary mice antibody of Caspase-12, CHOP and Bip/GRP78were added to the above membrane under gentle agitation for2h and sequentially incubated at4℃for overnight. After incubation, rinsing the membrane3times for15min in Tri-Buffered Saline (TBS) and then membrane was exposed to another horseradish peroxidase-coated secondary antibody for incubation at37℃for1h, and then the membrane was washed3times for15min with TBS. Following this, a chemiluminescent agent was applied to detect the reaction and an x-ay also was used to create image of antibodies bounding to the blot. The results were processed with film-developing and fixation and the images (grey-value) were analysed with gel documentation system to identify the protein expression of Caspase-12and Bip/GRP78in the substantia nigra among five groups.
(7) Statistical analysis:
All the data were expressed as mean±SD and data analysis was executed to using SAS package. One-way analysis of variance (ANOVA) was used to examine the statistically significant difference between the groups. A p value less than0.05was regarded as a statistically significant difference.
2. In-vitro experiment:effect of Anchanling on H2O2induced PC12cell apoptosis and endoplasmic reticulum stress
(1) Cell line and culture system
PC12as a cell line derived from mice pheochromocytoma was provided by the Shanghai Institute of Biochemistry and Cell Biology, CAS. The cell lines were placed on the pre-coated mice-tail collagen petri dish and grown by using DMEM culture media with10%fetal bovine serum and10%horse serum (Gibco BRL Company, USA), supplemented with2mmol/L glutamine,100U/ml streptomycin and100U/ml penicillin. All the cells were maintained in a humidified atmosphere of5%CO2incubation at37℃. Sub-culturing cells were performed once every other day and all the cells using in this experiment were at the stage of logarithmic phase and percentage of cell viability/survival rate were up to95%(Trypan blue stainning).
(2) Experimental groups and processes
In each experiment, PC cell lines were seeded into24-well plates (1.25×105cells/well concentration) and were cultured in DMEM for4h. Following this, the cultured cells were treated with different concentration of serum containing drug of ACL (50,100,200ug/ml) for3d before exposure to200mM H202. The extent level of cells apoptosis/death and cell viability were determined af ter12-hours' co-culture. The PC12cultured cells were assigned to control group, H202model group and ACL+H202group.
(3) Analysis of DNA ploidy on PC12cell
A total of100μl liquid from above sample were dyed in50mg/L propidium iodide (PI) and50mg/L RibonucleaseA and incubated in dark for30min. Next, all the data were acquired by flow cytometer and the results of subGl were measured in100percent. Every sample was used CellQuest to analyse10,000cells and the result was presented in percentage.
(4) Apoptosis cell immunofluorescence staining and fluorescent microscope assessing cell apoptosis in the substantia nigra
The culture medium containing apoptotic agent and other intervening substances were carefully removed from the seeded wells using a pipette and the cells were washed with1x D-PBS for1time. Followed by adding a solution (2mM Calcein AM and4mM Ethidium Homodier-1) to stain the cells (for24wells plate:adding to each well500μl; for12wells plate:adding to each well100μl; for6wells plate:adding to each well150μl) and incubating the cells at room temperature for30min. The makers indicating cells viability and death from stained cells were assessed by the fluorescent images captured under an inverted fluorescent microscope (Zeiss Axiovert135M, Mercury Lamp, Carl Zeis HBO100W/Z) equipped with excitation480nm filter and emission520nm filter
Results
1. Animal behavioural results
1.1PD model and behavioural results:
After administered with intraperitoneal injection of MPTP, The mice from PD model group were detected with various motor symptoms, such as tremor, pilo-erection and tail-suspension and these behaviors began from10-20min of last MTPT injection and lasted4-5hours and then declined, but symptoms of hypokinesia and bradykinesia still also appeared in this group. No statistical significant difference on the frequency of rotation was noted between the model group and ACL group before treatment (P>0.05). However, the frequency of apomorphine-induced rotations in the ACL group reduced gradually and there was statistically significant difference compared with model group at7d,14d and21d after last MTPT treatment (P<0.05, P<0.01, and P<0.01, respectively). Similarly, the results of hind-limb suspension test (HLS) indicated the HLS scores of model group and ACL group was not statistically significantly different from the control group (P>0,05and P>0.05) at1day before the last MPTP administration; In contrast, the HLS score of ACL group measuring at7d,14d, and21d after the MPTP injection was significant higher than the score of model group (P<0.01, P<0.01, and P<0.01, respectively). Similar scenario was seen on mice's pole-climbing behaviour. On the day before the last MPTP injection, the score of pole-climbing from model group and ACL group was found no statistically significant difference compared to the control group (P>0.05and P>0.05). However, pole-climbing behavior of model group was observed on7d,14d, and21d after last MTPT injection and the score significantly decreased (P<0.01, P<0.05and P<0.01, respectively) when comparing with the control group, which indicated the MPTP induced PD model was successful. The score of pole-climbing behavior from ACL group at14d and21d after last MPTP injury was significant increase from the model group (P<0.01and P<0.01).
1.2The results of immunohistochemistry, RT-PCR and Western blot
1.2.1Results of immunohistochemistry
Examination at21d after intraperitoneal injection of MPTP at the PD model group demonstrated a significant decrease in the number of TH-positive cells, GFAP-positive cells and OX-42-positive cells (P<0.01) while ACL group intragastrically injected with three different volume of ACL decoction (high-dose, median-dose, and low-dose, respectively) showed no significant reduce in number of TH-positive cells, GFAP-positive cells and OX-42-positive cells. However, the number of substantia nigra apoptosis cells in the ACL group was significantly reduced compared with PD model group (P<0.01). Meanwhile, the number of a-synuclein-positive cells, Parkin-positive cells and ubiquitin in the ACL group was significantly reduced compared with the PD model group (P<0.01).
1.2.2Results of RT-PCR analysis
The results indicated that intraperitoneally injection of MPTP could induce the stimulus of endoplasmic reticulum stress in the substantia nigra neurons in the mice and Anchanling, to some extent, could alleviate this stimulus.
1.2.3Results of Western blot analysis
Western blot analysis revealed that expression level of Caspase-12protein in the substantia nigra pars compacta (SNc) and CHOP protein was significantly elevated in the PD model group, which indicated the level of endoplasmic reticulum stress had been up-regulated. ACL group showed a significant decline in the expression level of Caspase-12protein in SNc and CHOP protein compared with the PD model group. GRP78/Bip expression level was significantly higher in the PD model group (P<0.05) than the level in the mice that did not undergo MPTP treatment in term of control group; moreover, GRP78/Bip expression level in the ACL group was significantly elevated compared to the PD model group (P<0.05).
2. Results of in-vitro experiment analysis
Under an electron microscope, the PC12cells (culture cells) of the control group had orderly arranged and adhered solidly to the surface of plastic petri dish, proliferated rapidly in long shuttle-like shape or prismatic shape and various lengths of axon-like pseudopodia emerged at the surface growing into an intertwined mesh pattern. In contrast, the cells from H202model group had disorderly arranged and loosely attached to the surface together with some length-lessen pseudopodia or absent pseudopodia as well as abundant amounts of suspension cells, disintegrated cells and dead cells. The morphological changes of culture cells from ACL+H202group were observed in an orderly arrangement together with few amounts of suspension cells and obvious pseudopodia formation, which was significant weaker than the H202model group. The cell viability in the100ug/ml ALC+H202group was significant higher than in the50ug/ml and150ug/ml ACL+H202group.
Compared with the control group, apoptosis index in the H202model group was showed a significant increase (P<0.01) and a similar trend was also seen on the number of a-synclein-positive cells with a significant increase (P<0.01) as well; however, the number of TH-positive cells in the model group was significant lower (P<0.01) than the number in the control group.
Compared to the H202model group, apoptosis index and the number of a-synuclein-positive cells in the ACL group were presented significantly reduce (P<0.01) but the amount of TH-positive cells were significantly higher than those in the model group (P<0.01).
Flow cytometer analysis revealed the results of the PC12cells DNA content, apoptotic cells cycles from H202model group were obviously seen shifting to the left compared wi th the control group; obvious subGl/GO of cells cycles appeared before G1/G0phase and the DNA index of subGl/GO was (94.50±21.3)%while the same phase percent in control group was (1.36±2.0)%; furthermore, cells percent distribution of PC12cells at G1/G0phase was lower in H202 group than in the control group; percentage of G2-phase and M-phase was diminished and DNA histogram presenting these two phase peak was nearly invisible. However, the ACLT+H202group, after incubating with different density of serum containing ACL (2.5%,5%and10%), PC12cells DNA index was (82.90±12.6)%,(69.34±15.7)%and (51.63±6.8)%, respectively.
Conclusions
ACL-treated mice's behaviour suggested that Parkinson's disease model induced by injecting with MPTP was successful; ACL could enhance the limb's motor coordination ability and ameliorate PD motor symptoms resulted from MPTP injury in PD mice model. The results of this study also indicated ESR and UPS might play pivotal role in the apoptosis of dopaminergic (DA) neurons and degradation of abnormal and/or un/misfolded protein via UPS pathway was involved in the early regulation process of ESR. The mutual promotion between ESR and UPS can maintain the dynamic balance of endoplasmic reticulum protein's synthesis and degradation, thereby to guarantee the cellular homeostasis stable.
Effects of Anchanling on the ESR and decreasing cytotoxic protein synthesis was accomplished by modulating protein pattern of the Caspase-12mRNA and intervening in the targeted expression in Caspase-12, CHOP and Bip/GRP78. Meanwhile, Anchanl ing affected and enhanced the UPS through intervening in the protein targeted expression of a-synuclein and ubiquitin, improving the ability of discovering and damaging these misfolded proteins, degrading/degenerating them and eliminating cytotoxic proteins so as to protect DA neurons cells in substantia nigra pars compacta and reducing accumulation of cells apoptosis. In addition, Anchanling could antagonise the neurons ESR and apoptotic stimuli induced by H2O2, thereby to protect the DA neuron cells. This was suggested that H202could induce cells apoptosis while Anchanling was able to suppress this progress of cells apoptosis. Also, intraperitoneally injecting MPTP could induce neurons ERS within substantia nigra in the C57BL/5J mice while ACL was able to significantly antagonise this stress of which antagonism was seen to be dose-dependence in a given range of ACL doses. Furthermore, ACL could inhibit cell apoptosis in the substantia nigra in PD mice model and enhance the function of UPS; the number of a-synuclein produced by endoplasmic reticulum was decreased and ubquitin degradation pathway became normal or had been improved. In conclusion, Anchanling can inhibit the cells apoptosis located in the substantia nigra and protect or improve functions of ERS and UPS in the MPTP-induced Parkinson's disease mice model.
帕金森病(Parkinson disease,PD)是渐行性神经退行性疾病,表现为黑质纹状体中多巴胺能神经元的进行性死亡,神经元胞浆内包涵体Lewy小体增多。PD的病因至今仍未完全明确,但公认的机制有兴奋性毒性和能量代谢紊乱、氧化应激、线粒体活性下降等功能紊乱。环境因素比如杀虫剂和其他因素也可以诱发散发性PD,而散发性PD占全部病例了90%以上。最近研究表明,PD疾病的发生与内质网应激和泛素-蛋白酶体系统有关。本课题结合国际帕金森病研究的最新进展和动态,采用腹腔注射MPTP诱导模拟人的PD慢性病程的动物模型,研究内质网应激与泛素-蛋白酶体系统与帕金森病发病的关系；同时进行天然中药新药安颤灵的药效学作用机理研究,为其进一步中药新药开发提供科学依据。
方法
1.动物实验：
(1)建立PD模型及实验分组：
选取健康雄性c57BL/5J小鼠60只(9周龄),随机分成5组,分别为PD模型组12只,予小鼠腹腔体内注射MPTP(30mg/kg.d),同时蒸溜水灌胃与安颤灵组等体积。正常对照组12只,蒸溜水灌胃与模型组相同,腹腔注射改为相同体积的生理盐水(30mg/kg.d)。安颤灵组(分高、中、低剂量组),每组各12只,该组在模型组基础上将灌胃的蒸溜水改为以上不同剂量的中药煎液。
(2)动物行为学观察：
注射后7d、14d和21d按0.25mg/kg腹腔注射阿扑吗啡,观察旋转行为的变化,共记录30mi n取平均值。同时注意观察小鼠震颤、活动迟缓、抓握、嗅探等异常行为。
(3)黑质区TH、GFAP.OX-42免疫组化检测：
注射后第21天,在2.5%戊巴比妥钠麻醉下,从左心室插管灌注4%多聚甲醛,之后断头取脑,经系列生化固定后。自近黑质部位起连续冠状切片,挑取对称的黑质区切片。行TH、GFAP、OX-42免疫组织化学染色,观察中脑黑质酪氨酸羟化酶(TH)阳性细胞,星形胶质细胞(GFAP)及小胶质细胞(OX-42)的变化,以了解DA能神经元损毁情况以及是否选择性地损毁多巴胺能神经元以及小胶质细胞的激活。切片再经系列生化免疫标记,置显微镜观察,计数阳性细胞数。
(4)a-synuelein、Parkin和泛素(ubiquitin)的免疫组化检测：
通过免疫组织化学染色法观察DA能神经元lewy小体的主要成分a-共核蛋白(a-synuelein)、Parkin和泛素(ubiquitin)的表达。切片经系列生化免疫标记后,显微镜观察,显微图象分析检测。模型组、正常组、安颤灵组处理时间和剂量完全相同。
(5)RT-PCR检测黑质区组织Caspase-12mRNA的表达：
GenBank检索小鼠Caspase-12序列,自行设计引物,由上海Sangon公司合成。Caspase-12引物(506bp)上游：5'-AGGCAACTCTCATGCAGTTC-3'(1319-1338bp),下游：5'-AACCATGTATGCCA GAG AC C-3'(1847-1866bp)。取总RNA4ul,加入Olig (dT)1ul,65℃15min,立即置冰上；加入10XRT缓冲液2μl,dNTP2ul,25mmol/L MgC122μl,RNA酶抑制剂1μl,A-MV1μl,加DEPC处理水至总体积20μl,42℃60min逆转录；取逆转录产物cDNA4μl,加入10%PCR缓冲液5μl,dNTP1μl, Caspase-12上游、下游引物各2μl(20pmol),Taq酶(5U/μl)0.25μ l,25mmol/LMgC123μl,加无菌双蒸水至总体积50μl。PCR条件：94℃5min,94℃40s,55℃40s,72℃90s,35个循环；72℃10min。取PCR扩增产物10μl行琼脂糖凝胶电泳,经凝胶成像系统扫描Caspase-12灰度值.。比较各组黑质区组织Caspase-12mRNA的表达。
(6)Western blot检测黑质区组织Caspase-12, CHOP、Bip/GRP78蛋白的表达：
50mg小鼠黑质组织用1%TritonX-100加蛋白酶抑制剂裂解,用牛血清白蛋白方法检测蛋白含量。每一样品取总蛋白30ug上样,用12%不连续聚丙烯酞胺凝胶电泳,转蛋白至硝酸纤维素膜；5%脱脂奶粉封闭2h；加进入Caspase-12、CHOP、Bip/GRP78第一抗体轻摇2h后4℃过夜；Tris磷酸盐缓冲液洗膜15min,共3次；加人辣根过氧化物酶标记的第二抗体37℃1h,Tris磷酸盐缓冲液洗膜15min,共3次。化学发光试剂作用后X胶片曝光,经显影、定影等处理后观察结果。用凝胶成像系统扫描图像,对其灰度分析,比较各组黑质区组织Caspase-12, Bip/GRP78蛋白的表达。
(7)数据统计：数据统计采用均值(x)±标准差(s),采用SAS软件包行单因素方差分析检验,以P<0.05表示有显著性差异。
2.离体实验：安颤灵对H2O2诱导PC12细胞凋亡及内质网应激的影响
(1)细胞株及培养体系：
Pc12细胞是小鼠肾上腺嗜铬细胞瘤细胞株,由中国科学院上海细胞生物学研究所提供。细胞接种在预先铺过鼠尾胶的培养皿中,用含有10%胎牛血清和10%马血清的DMEM培养基(美国Gibco BRL公司),同时加入2mmol/L谷氨酰胺、100u/ml链霉素和100u/ml青霉素,在温度37℃、5%CO2、饱和湿度的cO2培养箱中培养。隔天传代细胞1次,实验所用的细胞均处于对数生长期,且存活细胞百分率均在95%以上(台盼兰拒染法)。
(2)实验分组和处理：
在24孔培养板上,按每孔1．25×105个细胞的浓度加入Pc12,在DMEM中培养4h后,加入高、中、底不同浓度的安颤灵含药血清(50,100,200ug/ml)预处理3d,然后将细胞暴露与200mMH2O2中。经12h共同培养后,测定细胞的凋亡／死亡和存活情况。设正常和模型对照,实验重复3次。
(3)PC12细胞DNA倍体的分析：
在100μl样本液中加人含50mg/L碘化丙啶(美国BD公司)和50mg/L核糖核酸酶A(上海生工生物工程公司)的染液500μl,避光37℃孵育30min,流式细胞仪采集数据进行亚二倍体峰(subGl)的百分比分析。每份标本利用CellQuestTM软件分析10000个细胞,结果用百分比表示。
(4)凋亡细胞染色和荧光观察：
弃去介质和处理因素,用1×D-PBS轻轻清洗细胞一次。直接在细胞上添加适当量(约在24孔培养板上按50uL/孔,在12孔培养板上按100uL/孔或6孔培养板上按150uL/孔)的联合染液(2mM Calcein AM和4mM Ethidium Homodimer-1),在室温下孵育30分钟。标记的存活和死亡细胞用荧光显微系统(Zeiss Axiovert135M, Mercury Lamp, Carl Zeis HBO100W/Z),以485nm和590nm立即观察和拍照。
结果
1.动物实验结果：
1.1造模及行为学结果：腹腔注射MPTP后,PD模型组小鼠于第1次注药后10-20min均出现不同程度的震颤、竖毛、翘尾,上述症状持续4-5h后逐渐减轻,但仍有活动减少,动作迟缓等变化。治疗前模型组和安颤灵组组间的旋转圈数无显著性差异(P>0.05)。治疗后,实验第7天、第14天和第21天安颤灵组小鼠旋转次数逐渐减少,分别与模型组相比,差异显著(P<0.05、P<0.01、P<0.01)。悬挂行为结果显示：实验前1天(即首次注射MPTP前1天)观察小鼠悬挂行为,模型组、安颤灵组小鼠评分分别与对照组相比,无差异(P>0.05、P>0.05)；实验第7天、第14天和第21天观察模型组小鼠悬挂行为,其评分明显降低,分别与对照组相比,差异显著(P<0.01、P<0.05)；实验第14天和第21天安颤灵组小鼠悬挂实验评分明显升高,分别与模型组相比,差异显著(P<0.01、P<0.01)。实验前1天观察小鼠爬杆行为,模型组、安颤灵组小鼠评分分别与对照组相比,无差异(P>0.05、P>0.05)。注射MPTP第7天、第14天和第21天观察模型组小鼠爬杆行为,其评分明显降低,分别与对照组相比,差异显著(P<0.01、P<0.05、),提示造模成功。实验第14天和第21天安颤灵组评分明显升高,分别与模型组相比,差异显著(P<0.05、P<0.05)。
1.2.免疫组化、RT-PCR、Westernblot检测结果：
1.2.1免疫组化结果：PD模型组小鼠黑质区TH表达显著降低(P<0.01),GFAP、OX-42表达增多。经安颤灵灌胃处理的各组小鼠TH较模型组增多(P<0.01),而GFAP、OX-42阳性细胞数无明显变化。与模型组相比,中药安颤灵组a-synuclein.Parkin、ubiquitin阳性细胞数均减少(P<0.01)。
1.2.2RT-PCR结果提示,腹腔注射MPTP可诱导小鼠脑黑质区神经元的内质网应激反应,而安颤灵对这一应激反应有一定的缓解作用。
1.2.3.Western blot检测结果显示：模型组小鼠黑质区Caspase-12、CHOP蛋白表达水平较正常对照组显著上调(P<0.01),提示黑质区神经元的内质网应激反应提高。安颤灵组Caspase-12、CHOP蛋白表达水平较模型组明显降低(P<0.05)。模型组小鼠黑质区组织GRP78/Bip蛋白的表达较正常对照组上调,安颤灵组GRP78/Bip蛋白的表达较模型组进一步上调(P<0.05)。
2.离体实验结果：
实验光镜下观察发现,对照组PC12细胞排列整齐,贴壁生长旺盛,形态规则,呈长梭形或长棱形,有长短不等的类似神经轴突的伪足,交织成网状。H2O2损伤后的模型组细胞,排列紊乱,贴壁不牢,伪足缩短火消失,大量细胞悬浮,碎裂,死亡。安颤灵含药血清培养细胞变化较模型组明显减弱。细胞排列尚整齐,悬浮细胞较少,可见明显伪足。与大、小剂量组相比,中剂量组细胞的存活率显著升高(P<0.01)；与正常组相比,模型组细胞凋亡指数明显增加(P<0.01),a-synuclein、阳性细胞数亦均增多,而TH阳性细胞明显减少；与H2O2组相比,安颤灵组细胞凋亡指数明显降低,a-synuclein阳性细胞数均减少,TH阳性细胞明显增加(P<0.01)。H2O2模型组PC12细胞与正常对照组相比细胞周期明显左移。在细胞G1/Go期前有明显的亚二倍体峰(subGl-Go),处于此期的细胞比率为(94.50±21.3)%,而正常对照组处于此期的细胞比率为(1.36±2.0)%。而且H2O2模型组G1/G0期及所占的比率明显低于正常对照组,G2期及M期的细胞所占的比例也减少,在细胞周期图中代表这两期的峰几乎不可见。而同时给予不同浓度安颤灵含药血清(2.5%、5%、10%)孵育后,Pc12细胞subG1-Go百分比分别为(82.90±12.6)%、(69.34±15.7)%、(51.63±6.8)%。
结论
中药安颤灵小鼠行为学表明,采用MPTP注射法,造模成功；安颤灵能提高肢体运动协调能力,对MPTP诱导所致的PD模型小鼠PD运动体征具有明显的改善作用。ESR和UPS可能是PD多巴胺能神经元凋亡的关键因素,内质网应激的早期调节过程就存有泛素-蛋白酶体系统对异常蛋白的降解活动。ERS和UPS有相互促进作用,以达到内质网蛋白合成与降解的动态平衡,保证内环境的稳定。中药安颤灵就是通过黑质区组织Caspase-12mRNA的表达和Caspase-12, CHOP、Bip-GRP78蛋白的表达靶点的干预来实现对内质网应激反应(ESR)影响和改善,减少细胞毒性蛋白的合成。通过a-synuelein.和泛素(ubiquitin)蛋白的表达靶点的干预来实现对UPS的影响和改善,提高发现和破坏这些装配和折叠错误的蛋白的能力,使它们及时降解,消除细胞毒性蛋白,达到改善保护黑质区DA能神经元神经细胞,减少细胞凋亡。同时,对H202诱导的神经元ERS与凋亡反应具有显著地拮抗作用,从而达到保护细胞的作用。提示,H2O2能诱导细胞凋亡,而安颤灵可抑制这一细胞凋亡过程。腹腔注射MPTP可诱导c57小鼠黑质区神经元的内ESR,而安颤灵对这一应激反应具有明显拮抗作用,且该作用在一定浓度范围内呈现剂量依赖性。安颤灵可抑制PD小鼠模型黑质细胞的凋亡,并可改善UPS的功能。表明细胞内质网生成的毒性蛋白质a-synuclein.减少,毒性蛋白质的泛素降解通路正常或提高。表明ERS功能受到保护干预和UPS的功能活性良好。提示安颤灵可抑制PD小鼠模型黑质细胞的凋亡,可保护和改善ERS及UPS的功能。
Objectives
Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of the dopamine (DA) neurons in the substantia nigra pars compacta; accumulation of eosinophilic intraneural inclusions called Lewy bodies in the brain. Etiology of cell death still remains unknown, but it may result from generation of excitotoxicity, metabolic disorder, free radicals, oxidative stress and mitochondrial abnormality; also, environmental factors, such as exposure to the pesticides, has long been suggested as a risk factor for sporadic PD of which account for up to90%of all cases of PD. Ongoing researches suggest that the cause of PD was associated with the endoplasmic reticulum stress (ERS) and Ubiquitin-Protea some System (UPS). Based on these findings, together with the latest international studies, this present study aimed to investigate the relationship between ERS-UPS and etiology of PD and to elucidate an effect of Anchanling (a Chinese medicine, used as clinical therapy for early stage PD) on the ERS-UPS in PD mice model through establishing a mouse PD model induced by intraperitoneal injection of MPTP; furthermore the study was to explore the pharmacodynamic mechanism of Anchanling and to provide the development of new Chinese medicine for a treatment for PD with more scientific evidence.
Materials and Methods
1. The animal experiment:
(1) PD model establishment and experimental groups:
A total of60healthy male C57BL/5J mice (9-week-old) were selected and then was randomly divided into5groups:the PD model group (12), control group (12) and ACL group (high-dose, medium-dose and low-dose; every group contained12mice). At the start of experiment, the mice from PD model group were intraperitoneally injected with MPTP (30mg/kg. d) and were administered intragastrically perfusion with distilled water; The control group was intraperitoneally injected with normal saline (30mg/kg. d) and intragastrical method was the same as in the PD model group; three sub-group of ACL group were injected with the same volume of MPTP as did in the PD model group and then administrated intragastrically with three different volume of ALC decoction (high-dose, medium-dose and low-dose, respectively).
(2) Animal behaviour observation:
All the mice were intraperitoneally injected with0.25mg/kg apomorphine every seven days from the beginning of experiment postoperatively and induced rotational behaviour were observed for30minutes after injection and the rotations were recorded for three weeks (7d,14d and21d). Meanwhile, other abnormal behaviours, such as tremor, bradykinesia, grasping and sniffing were observed as well.
(3) Immunohistochemistry test on Substantia nigra tyrosin hytroxylase (TH), GFAP and OX-42:
Three weeks after the administration, the mice were anesthetized by intraperitoneal injection of2.5%pentobarbital, followed by perfusion of4%papraformaldehyde via left ventricular incubation. Brains were harvested and positioned in a stereotaxic apparatus. Serial coronal sections were prepared originating from the substantia nigra using freezing microtome and the symmetrical sections from the substantia nigra were selected for subsequent assay. Immunohistochemistry staining on TH, GFAP and OX-42was applied to observe the morphological change occurring among TH-positive cells, GFAP and OX-42so as to investigate damage level of dopaminergic (DA) neurons and to recognise whether GFAP selectively attack DA neurons as well as to identify whether OX-42has been activated. The sections were reused for immunohistochemical labeling and the numbers of TH-positive cells were counted under the fluorescent microscope.
(4) Immunohistochemistry test on a-synuclein, Parkin and ubiquitin:
a-synuclein, Parkin, and ubiquin, as the main components of Lewy bodies, their protein expression could be observed via immunohistochemical staining. After a serial of immunohistochemical processes (such as rinse, blocking and makers) sections were placed under the fluorescent microscope to count the above three proteins positive cells and then assay via microimage analysis. All the procedures including the doses of reagents and time consuming applying in the PD model group, control group and ACL group were the same.
(5) RT-PCR assaying the expression of substantia nigra tissue Caspase-12mRNA
The nucleotide sequences were based on the published Caspase-12(retrieved GenBank) and the self-designed primers was synthesized by Shanghai Sangon Company. Caspase-12primer (506bp) displayed as following, forward:5'AGGC AACTCTCATGCAGTTC-3'(1319-1338bp), reverse:5'-AACCATGTATGCCAGAG AC C-3'(1847-1866bp). First-strand complementary DNA was synthesised as following:taking4ul total RNA and adding withlu101ig (dT) at65℃for15min, then immediately placing on the ice; adding2μl10xRT buffer solution,2ul dNTP,2μl125mmol/LMgCl,1μ L ribonulclease inhibitors,1μl L A-MV, and adding DEPC-treated water to make total volume reach20μl and then keeping60min at42℃for reverse transcription; The PCR mixture contained4μl cDNA,5μl10%PCR buffer solution,1μ1dNTP,2μl (20pmol) forward primers of Caspase-12and2μl (20pmol) reverse primers of Caspase-12,0.25UlTaq DNA polymerase (5U/μl),3μl125mmol/L MgC12, and sterile double-distilled water in a final volume50μl was also prepared. The procedure of PCR was performed at94℃for5min, followed by35cycles at94℃for40s,55℃for40s,72℃for90s and extension at72℃for10min. Next,10μl PCR products were electrophoresed on agarose gel and the gels image were examined and analysed by gel documentary system to detect Caspase-12grey value. Finally, to compare the Caspase-12mRNA expressions among PD model group, control group and ACL group.
(6) Western Blotting analysis of Caspase-12, CHOP, Bip/GRP78
50mg mice substantia nigra tissue was employed with1%TritonX-100and protease inhibitors to encourage cleaves of the cells, with bovine serum albumin (BSA) to detect protein concentrations of cells lysates. An equal amount of protein for each sample (30ug in total) were separated by using12%sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with5%skimmed milk powder for2h. After blocking, the primary mice antibody of Caspase-12, CHOP and Bip/GRP78were added to the above membrane under gentle agitation for2h and sequentially incubated at4℃for overnight. After incubation, rinsing the membrane3times for15min in Tri-Buffered Saline (TBS) and then membrane was exposed to another horseradish peroxidase-coated secondary antibody for incubation at37℃for1h, and then the membrane was washed3times for15min with TBS. Following this, a chemiluminescent agent was applied to detect the reaction and an x-ay also was used to create image of antibodies bounding to the blot. The results were processed with film-developing and fixation and the images (grey-value) were analysed with gel documentation system to identify the protein expression of Caspase-12and Bip/GRP78in the substantia nigra among five groups.
(7) Statistical analysis:
All the data were expressed as mean±SD and data analysis was executed to using SAS package. One-way analysis of variance (ANOVA) was used to examine the statistically significant difference between the groups. A p value less than0.05was regarded as a statistically significant difference.
2. In-vitro experiment:effect of Anchanling on H2O2induced PC12cell apoptosis and endoplasmic reticulum stress
(1) Cell line and culture system
PC12as a cell line derived from mice pheochromocytoma was provided by the Shanghai Institute of Biochemistry and Cell Biology, CAS. The cell lines were placed on the pre-coated mice-tail collagen petri dish and grown by using DMEM culture media with10%fetal bovine serum and10%horse serum (Gibco BRL Company, USA), supplemented with2mmol/L glutamine,100U/ml streptomycin and100U/ml penicillin. All the cells were maintained in a humidified atmosphere of5%CO2incubation at37℃. Sub-culturing cells were performed once every other day and all the cells using in this experiment were at the stage of logarithmic phase and percentage of cell viability/survival rate were up to95%(Trypan blue stainning).
(2) Experimental groups and processes
In each experiment, PC cell lines were seeded into24-well plates (1.25×105cells/well concentration) and were cultured in DMEM for4h. Following this, the cultured cells were treated with different concentration of serum containing drug of ACL (50,100,200ug/ml) for3d before exposure to200mM H202. The extent level of cells apoptosis/death and cell viability were determined af ter12-hours' co-culture. The PC12cultured cells were assigned to control group, H202model group and ACL+H202group.
(3) Analysis of DNA ploidy on PC12cell
A total of100μl liquid from above sample were dyed in50mg/L propidium iodide (PI) and50mg/L RibonucleaseA and incubated in dark for30min. Next, all the data were acquired by flow cytometer and the results of subGl were measured in100percent. Every sample was used CellQuest to analyse10,000cells and the result was presented in percentage.
(4) Apoptosis cell immunofluorescence staining and fluorescent microscope assessing cell apoptosis in the substantia nigra
The culture medium containing apoptotic agent and other intervening substances were carefully removed from the seeded wells using a pipette and the cells were washed with1x D-PBS for1time. Followed by adding a solution (2mM Calcein AM and4mM Ethidium Homodier-1) to stain the cells (for24wells plate:adding to each well500μl; for12wells plate:adding to each well100μl; for6wells plate:adding to each well150μl) and incubating the cells at room temperature for30min. The makers indicating cells viability and death from stained cells were assessed by the fluorescent images captured under an inverted fluorescent microscope (Zeiss Axiovert135M, Mercury Lamp, Carl Zeis HBO100W/Z) equipped with excitation480nm filter and emission520nm filter
Results
1. Animal behavioural results
1.1PD model and behavioural results:
After administered with intraperitoneal injection of MPTP, The mice from PD model group were detected with various motor symptoms, such as tremor, pilo-erection and tail-suspension and these behaviors began from10-20min of last MTPT injection and lasted4-5hours and then declined, but symptoms of hypokinesia and bradykinesia still also appeared in this group. No statistical significant difference on the frequency of rotation was noted between the model group and ACL group before treatment (P>0.05). However, the frequency of apomorphine-induced rotations in the ACL group reduced gradually and there was statistically significant difference compared with model group at7d,14d and21d after last MTPT treatment (P<0.05, P<0.01, and P<0.01, respectively). Similarly, the results of hind-limb suspension test (HLS) indicated the HLS scores of model group and ACL group was not statistically significantly different from the control group (P>0,05and P>0.05) at1day before the last MPTP administration; In contrast, the HLS score of ACL group measuring at7d,14d, and21d after the MPTP injection was significant higher than the score of model group (P<0.01, P<0.01, and P<0.01, respectively). Similar scenario was seen on mice's pole-climbing behaviour. On the day before the last MPTP injection, the score of pole-climbing from model group and ACL group was found no statistically significant difference compared to the control group (P>0.05and P>0.05). However, pole-climbing behavior of model group was observed on7d,14d, and21d after last MTPT injection and the score significantly decreased (P<0.01, P<0.05and P<0.01, respectively) when comparing with the control group, which indicated the MPTP induced PD model was successful. The score of pole-climbing behavior from ACL group at14d and21d after last MPTP injury was significant increase from the model group (P<0.01and P<0.01).
1.2The results of immunohistochemistry, RT-PCR and Western blot
1.2.1Results of immunohistochemistry
Examination at21d after intraperitoneal injection of MPTP at the PD model group demonstrated a significant decrease in the number of TH-positive cells, GFAP-positive cells and OX-42-positive cells (P<0.01) while ACL group intragastrically injected with three different volume of ACL decoction (high-dose, median-dose, and low-dose, respectively) showed no significant reduce in number of TH-positive cells, GFAP-positive cells and OX-42-positive cells. However, the number of substantia nigra apoptosis cells in the ACL group was significantly reduced compared with PD model group (P<0.01). Meanwhile, the number of a-synuclein-positive cells, Parkin-positive cells and ubiquitin in the ACL group was significantly reduced compared with the PD model group (P<0.01).
1.2.2Results of RT-PCR analysis
The results indicated that intraperitoneally injection of MPTP could induce the stimulus of endoplasmic reticulum stress in the substantia nigra neurons in the mice and Anchanling, to some extent, could alleviate this stimulus.
1.2.3Results of Western blot analysis
Western blot analysis revealed that expression level of Caspase-12protein in the substantia nigra pars compacta (SNc) and CHOP protein was significantly elevated in the PD model group, which indicated the level of endoplasmic reticulum stress had been up-regulated. ACL group showed a significant decline in the expression level of Caspase-12protein in SNc and CHOP protein compared with the PD model group. GRP78/Bip expression level was significantly higher in the PD model group (P<0.05) than the level in the mice that did not undergo MPTP treatment in term of control group; moreover, GRP78/Bip expression level in the ACL group was significantly elevated compared to the PD model group (P<0.05).
2. Results of in-vitro experiment analysis
Under an electron microscope, the PC12cells (culture cells) of the control group had orderly arranged and adhered solidly to the surface of plastic petri dish, proliferated rapidly in long shuttle-like shape or prismatic shape and various lengths of axon-like pseudopodia emerged at the surface growing into an intertwined mesh pattern. In contrast, the cells from H202model group had disorderly arranged and loosely attached to the surface together with some length-lessen pseudopodia or absent pseudopodia as well as abundant amounts of suspension cells, disintegrated cells and dead cells. The morphological changes of culture cells from ACL+H202group were observed in an orderly arrangement together with few amounts of suspension cells and obvious pseudopodia formation, which was significant weaker than the H202model group. The cell viability in the100ug/ml ALC+H202group was significant higher than in the50ug/ml and150ug/ml ACL+H202group.
Compared with the control group, apoptosis index in the H202model group was showed a significant increase (P<0.01) and a similar trend was also seen on the number of a-synclein-positive cells with a significant increase (P<0.01) as well; however, the number of TH-positive cells in the model group was significant lower (P<0.01) than the number in the control group.
Compared to the H202model group, apoptosis index and the number of a-synuclein-positive cells in the ACL group were presented significantly reduce (P<0.01) but the amount of TH-positive cells were significantly higher than those in the model group (P<0.01).
Flow cytometer analysis revealed the results of the PC12cells DNA content, apoptotic cells cycles from H202model group were obviously seen shifting to the left compared wi th the control group; obvious subGl/GO of cells cycles appeared before G1/G0phase and the DNA index of subGl/GO was (94.50±21.3)%while the same phase percent in control group was (1.36±2.0)%; furthermore, cells percent distribution of PC12cells at G1/G0phase was lower in H202 group than in the control group; percentage of G2-phase and M-phase was diminished and DNA histogram presenting these two phase peak was nearly invisible. However, the ACLT+H202group, after incubating with different density of serum containing ACL (2.5%,5%and10%), PC12cells DNA index was (82.90±12.6)%,(69.34±15.7)%and (51.63±6.8)%, respectively.
Conclusions
ACL-treated mice's behaviour suggested that Parkinson's disease model induced by injecting with MPTP was successful; ACL could enhance the limb's motor coordination ability and ameliorate PD motor symptoms resulted from MPTP injury in PD mice model. The results of this study also indicated ESR and UPS might play pivotal role in the apoptosis of dopaminergic (DA) neurons and degradation of abnormal and/or un/misfolded protein via UPS pathway was involved in the early regulation process of ESR. The mutual promotion between ESR and UPS can maintain the dynamic balance of endoplasmic reticulum protein's synthesis and degradation, thereby to guarantee the cellular homeostasis stable.
Effects of Anchanling on the ESR and decreasing cytotoxic protein synthesis was accomplished by modulating protein pattern of the Caspase-12mRNA and intervening in the targeted expression in Caspase-12, CHOP and Bip/GRP78. Meanwhile, Anchanl ing affected and enhanced the UPS through intervening in the protein targeted expression of a-synuclein and ubiquitin, improving the ability of discovering and damaging these misfolded proteins, degrading/degenerating them and eliminating cytotoxic proteins so as to protect DA neurons cells in substantia nigra pars compacta and reducing accumulation of cells apoptosis. In addition, Anchanling could antagonise the neurons ESR and apoptotic stimuli induced by H2O2, thereby to protect the DA neuron cells. This was suggested that H202could induce cells apoptosis while Anchanling was able to suppress this progress of cells apoptosis. Also, intraperitoneally injecting MPTP could induce neurons ERS within substantia nigra in the C57BL/5J mice while ACL was able to significantly antagonise this stress of which antagonism was seen to be dose-dependence in a given range of ACL doses. Furthermore, ACL could inhibit cell apoptosis in the substantia nigra in PD mice model and enhance the function of UPS; the number of a-synuclein produced by endoplasmic reticulum was decreased and ubquitin degradation pathway became normal or had been improved. In conclusion, Anchanling can inhibit the cells apoptosis located in the substantia nigra and protect or improve functions of ERS and UPS in the MPTP-induced Parkinson's disease mice model.
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