Effect Of Anchanling On Endoplasmic Recticulum Stress And Ubiquitin-Proteasome System In Parkinsons Disease Mice Model

ObjectivesParkinson’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 Methods1. 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-12mRNAThe 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/GRP7850mg 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 processesIn 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 PC12cellA 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 nigraThe 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 filterResults1. Animal behavioural results1.1PD 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 blot1.2.1Results of immunohistochemistryExamination 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 analysisThe 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 analysisWestern 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 analysisUnder 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.ConclusionsACL-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.