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Toxicology Letters 222 2013 155 163 Contents lists available at ScienceDirect Toxicology Letters jou rn al hom epage Neuroprotection by tetrahydroxystilbene glucoside in the MPTP mouse model of Parkinson s disease Lingling Zhangb LinHong Huangb Liangwei Chenc Dingjun Haob Jianzong Chena aResearch Center of Traditional Chinese Medicine Xijing Hospital Fourth Military Medical University Xi an 710032 China bDepartment of Scientifi c Research Hong Hui Hospital Xi an Jiaotong University College of Medicine Xi an 710054 China cInstitute of Neurosciences Fourth Military Medical University Xi an 710032 China h i g h l i g h t s TSG promoted dopamine neuron survival in MPTP treated mouse model of PD The PI3K Akt signaling pathway may have mediated the protection of TSG against MPTP TSG has potential as a naturally derived treatment to halt the progression of PD a r t i c l e i n f o Article history Received 14 June 2013 Received in revised form 19 July 2013 Accepted 24 July 2013 Available online 1 August 2013 Keywords Parkinson s disease Tetrahydroxystilbene glucoside Neuroprotection Tyrosine hydroxylase Phosphatidylinositol 3 kinase PI3K Akt a b s t r a c t Our in vitro experiments suggested that tetrahydroxystilbene glucoside TSG affords a signifi cant neu roprotective effect against MPP induced damage and apoptosis in PC12 cells though activation of the PI3K Akt pathway This study was aimed to investigate the potential neuroprotective effect of TSG in 1 methyl 4 phenyl 1 2 3 6 tetrahydropypridine MPTP treated mouse model of Parkinson s disease PD We found that treatment of TSG protected dopaminergic neurons by preventing MPTP induced decreases in substantia nigra tyrosine hydroxylase TH positive cells and striatal dopaminergic transporter DAT protein levels Furthermore it was also associated with increasing striatal Akt and GSK3 phosphoryla tion up regulation of the Bcl 2 BAD ratio and inhibition of the activation of caspase 9 and caspase 3 These results showed that TSG promoted dopamine neuron survival in vivo the PI3K Akt signaling pathway may have mediated the protection of TSG against MPTP suggesting that TSG treatment might represent a neuroprotective treatment for PD Crown Copyright 2013 Published by Elsevier Ireland Ltd All rights reserved 1 Introduction Parkinson s disease PD is the second most common neurode generative disorder It is characterized clinically by a variety of motor dysfunctions such as resting tremor bradykinesia rigid ity and postural instability These symptoms are attributed to the reduction in striatal dopamine DA level which results from the selective and progressive degeneration of dopaminergic DAergic neurons in the substantia nigra pars compacta SNpc Wakamatsu et al 2008 Moore et al 2005 Lees et al 2009 The causes of PD are still unclear however several studies suggest the involve ment of oxidative stress mitochondria dysfunction and apoptosis Olanow and Tatton 1999 Lin and Beal 2006 Levy et al 2009 Corresponding author Tel 86 29 87800002 fax 86 29 87894724 Corresponding author Tel 86 29 84775955 fax 86 29 82555145 E mail addresses haodingjundoctor D Hao chenjz57 jzchen57 J Chen Current PD medications treat the symptoms of the disease without halting or delaying the degeneration of DAergic neurons Toulouse and Sullivan 2008 There has been considerable inter est in neuroprotection as a therapeutic strategy for PD Yacoubian and Standaert 2009 Serine threonine kinase Akt is a pro survival kinase that is activated by the phosphorylation at the phosphoinosi tide 3 kinases PI3K Akt pathway which promotes cell survival and prevents apoptosis Dudek et al 1997 Some studies have shown that phosphoSer473 Akt is predominantly down regulated in melanized neurons from the SNpc of patients with PD obtained at autopsy Timmons et al 2009 Malagelada et al 2008 Many reports using cellular and animal models of PD have implicated Akt activation in the neuroprotection of DAergic neurons D Astous et al 2006 Fukui et al 2010 Nair and Olanow 2008 Ries et al 2006 Sagi et al 2007 Weinreb et al 2009 Yu et al 2008 Therefore the activation of PI3K Akt represents a promising new approach for the prevention and treatment of PD Polygonum multifl orum thunb PM is a medicinal herb that has long been used as a constituent of traditional Chinese prescrip tions aimed at treating age related impairments such as cognitive 0378 4274 see front matter Crown Copyright 2013 Published by Elsevier Ireland Ltd All rights reserved http dx doi org 10 1016 j toxlet 2013 07 020 156L Zhang et al Toxicology Letters 222 2013 155 163 Fig 1 Chemical structure of TSG 2 3 5 4 tetrahydroxystilbene 2 O d glucoside defi ciencies Chan et al 2003 Um et al 2006 Chan et al 2002 PM extract PME has been reported to have neuroprotective effects and was associated with behavioral improvement in a mouse model of paraquat and maneb induced DAergic neuronal damage Li et al 2005 One of the main active ingredients of PM is 2 3 5 4 tetrahydroxystilbene 2 O d glucoside TSG Fig 1 it exhibits antioxidative anti infl ammatory and inhibiting oxidative stress effects Li et al 2010 Ryu et al 2002 Wang et al 2008 2009 We have also recently found TSG to be protective against MPP induced toxicity in PC12 cells through its antioxidant activity and the modulation of PI3K Akt pathways Qin et al 2011 However to date the mechanisms that underlie the protection effect of TSG on the neurotoxin have not been fully understood in vivo MPTP or 1 methyl 4 phenyl 1 2 3 6 tetrahydropypridine can selectively damage neurons in the nigrostriatal dopaminergic path way and cause Parkinsonism in humans nonhuman primates and mice mice have therefore become well accepted as a model for PD Kopin and Markey 1988 Langston 1996 Betarbet et al 2002 Gr nblatt et al 2000 The neurotoxic effects of MPTP are thought to be mediated by 1 methyl 4 phenyl pyridinium MPP an oxidative product of MPTP converted by monoamine oxidase B MAO B MPP has a high affi nity for the dopamine transporter DAT and causes mitochondrial dysfunction and oxidative stress Heikkila et al 1984 Javitch et al 1985 Kim Han et al 2011 Tipton and Singer 1993 The C57BL 6 mouse strain is known to be highly susceptible to the neurotoxin and MPTP treated mice have been widely used as an in vivo model to test therapeutic strategies in PD Mori et al 2005 In the present experiments we investigated the effects of TSG on behavioral and neurohistological alterations in mice caused by treatment with MPTP Moreover we sought to determine whether neuroprotection by TSG against MPTP is mediated by the activation of the PI3K Akt pathway 2 Materials and methods 2 1 Reagents TSG HPLC 98 was obtained from the Sichuan Weikeqi Biological Technol ogy Co Ltd Sichuan China MPTP and rabbit anti Tyrosine Hydroxylase TH polyclonal antibody were obtained from Sigma Rat anti dopamine transporter DAT monoclonal antibody was obtained from Chemicon Alexa Fluor 594 donkey anti rabbit IgG H L were obtained from the Invitrogen Company Rabbit anti Akt total antibody rabbit anti phospho Akt Ser473 antibody rabbit anti GSK3 total and rabbit anti phospho GSK3 Ser9 antibody and the rabbit anti cleaved caspase 3 antibody were purchased from Cell Signaling Technology Rabbit anti Bcl 2 anti BAD antibody and actin were from Santa Cruz Biotechnology Mouse anti caspase 9 and Horseradish peroxidase HRP conjugated anti rabbit anti rat and anti mouse antibodies were obtained from the Beyotime Institute of Biotech nology Shanghai China 2 2 Animals and treatment protocol All procedures were approved by the Animal Ethical Committee of Xijing Hos pital Fourth Military Medical University and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals Male C57BL 6 mice 23 26 g Fourth Military Medical University Experimental Animal Center China were maintained under standard housing conditions with constant temperature Food and water were available ad libitum The animals were divided into fi ve groups with 10 in each The effects of TSG on MPTP induced parkinsonism were studied in the following experimental groups A NS saline i p NS i g B MPTP i p NS i g C MPTP i p TSG 20 mg kg day i g D MPTP i p TSG 40 mg kg day i g and E NS i p TSG 40 mg kg day i g Animals in groups A E received saline or MPTP 30 mg kg i p daily for 7 days vehicle saline or TSG treatment i g was started 1 h after the last MPTP administration and given daily for 14 days Mice were killed by cervical dis location or perfusion after the last vehicle or TSG administration and behavior observation 2 3 Pole test The pole test was performed according to previously published methods Matsuura et al 1997 Briefl y the animals were positioned head upward near the top of a rough surfaced iron pole 10 mm in diameter and 55 cm in height The time taken until they turned completely downward defi ned as Tturn and the time taken to climb down to the fl oor TLA were recorded as indices of motor coordi nation defi cit Performance in the pole test was measured 14 days after the last injection of MPTP This test was conducted 1 h after the administration of the test drugs Fig 2 The pole test performance of mice pretreated with MPTP with or without TSG The pole test was conducted as described in the text and the time taken for mice to turn completely downward Tturn A and the time taken to climb to the fl oor TLA B were determined Each column represents the means SEM n 10 each P 0 01 vs NS NS group P 0 05 P 0 01 vs MPTP NS group L Zhang et al Toxicology Letters 222 2013 155 163157 Fig 3 Distribution of TH positive neurons in the substantia nigra pars compacta of distinct animal groups A and B Representative photomicrographs of TH immunoreactive neurons in the substantia nigral compacts SNpc The White arrow pointed to typical TH positive neurons with nuclei Higher magnifi cation of the area in the rectangle is shown in C Representative images are shown from one of three independent experiments shown Data are expressed as means SEM n 10 per group Bar 200 m P 0 01 vs NS NS group P 0 05 P 0 01 vs MPTP NS group 2 4 Perfusion and tissue processing At the end of the experiment half of the animals in each group n 10 were sacrifi ced under 1 pentobarbital sodium 100 mg kg and then perfused via intrac ardial infusion with 0 9 saline followed by 4 paraformaldehyde PFA pH 7 4 After intracardial perfusion the brains were collected and post fi xed in 4 PFA for 24 h at 4 C and cut into 30 m coronal sections encompassing the entire SNpc antero posterior levels Bregma 2 80 mm to 3 64 mm with the aid of the mouse brain atlas Franklin and Paxinos 1997 The other ten animals in each group were killed by cervical dislocation and the tissue of their ventral midbrain was dissected rapidly on ice Jackson Lewis and Przedborski 2007 frozen in liquid nitrogen and stored at 80 C until use 2 5 Immunofl uorescence histochemistry Immunofl uorescence histochemistry of brain tissues was performed as described in previous reports with minor modifi cations Wang et al 2007 For double staining experiments the slides were washed at least three times with 0 01 M PBS for 5 min They were then incubated at 4 C overnight with rabbit anti TH antibody 1 1000 in 0 01 M PBS containing 3 bovine serum albumin and 0 2 Tri ton X 100 On the second day the slides were washed three times with 0 01 M PBS for 5 min and were then treated with donkey anti rabbit IgG labeled with FITC for 3 h The sections were washed mounted on coverslips and then analyzed by inverted microscope IX51 12PH Olympus at a magnifi cation of 200 TH immunoreactive neurons were counted as previously described Jackson Lewis et al 1995 Data from the experiment were processed by statistical analysis 2 6 Western blot For Western blot analysis isolated tissues from the ventral midbrain were rinsed twice with cold PBS and homogenized in lysis buffer that contained proteinase inhibitors Lysates were cleared by centrifugation at 12 000 rpm at 4 C for 5 min and the total protein content in the supernatant was determined using a BCA Protein Assay Kit The sample was then boiled for 5 min and 10 l containing 40 g of pro tein of each sample was applied to a sodium dodecyl sulfate SDS polyacrylamide 12 gel for electrophoresis The gel was run at 80 V for 1 h and 120 V for 2 h and then electrophoretically transferred to polyvinylidene difl uoride PVDF membranes Pierce Co Ltd at 150 V for 1 h The membranes were blocked for 2 h with 5 BSA in Tris buffered saline TBS containing 0 05 Tween 20 TBST at 37 C They were then incubated at 4 C overnight with the respective primary antibodies anti DAT anti p Akt anti Akt anti p GSK3 anti GSK3 anti BAD anti caspase 9 which contains 47 kDa and 35 kDa specifi cities for assaying pro and cleaved caspase 9 actin 1 1000 anti Bcl 2 anti cleaved caspase 3 which contains 17 19 kDa specifi cities for assaying cleaved caspase 3 1 200 After rinsing fi ve times in TBST for 5 min membranes were incubated for 2 h at 37 C with anti rabbit or anti mouse or anti rat IgG peroxidase conjugated 1 1000 Immunoreactivity was visualized by an Enhanced Chemiluminescence ECL fl uores cence detection system GE USA The blots were visualized with ECL plus reagent and the results were analyzed using Lab Image version 2 7 1 Kapelan GmbH Halle Germany For DAT Bcl 2 BAD GSK3 and Akt the density of each band was nor malized to its respective loading control actin For p Akt and p GSK3 the total levels of the kinase Akt or GSK3 were used for normalization 158L Zhang et al Toxicology Letters 222 2013 155 163 Fig 4 Treatment of TSG restored the reduction of DAT expression in the striatum of MPTP treated mice A The decrease of DAT expression was restored by TSG treatment B Histogram representing the quantitative analysis of DAT level normalized to actin protein respectively Data are expressed as means SEM n 10 per group P 0 05 P 0 01 vs NS NS group P 0 01 vs MPTP NS group 2 7 Stereology TH positive SNc neurons were counted from a mouse in either group by an optical fractionator as described previously West 1999 Double blinded counting was performed manually 2 8 Statistical analysis Measurement data were expressed as means standard error of the mean SEM Comparison of more than two groups was performed by analysis of variance ANOVA with post hoc Tukey s tests Differences were considered signifi cant when P 0 05 All results represent at least three independent experiments performed in triplicate Data analyses were performed with SPSS 16 0 software SPSS Chicago IL USA 3 Results 3 1 The pole test In the pole test the animals that had received repeated treat ment with MPTP showed signifi cantly prolonged Tturn P 0 01 and TLA P 0 01 compared to the control animals that had received saline alone during the same period indicating an induction of bradykinesia Pretreatment of TSG 20 or 40 mg kg i g signifi cantly reversed the MPTP induced prolongation of Tturn P 0 05 and TLA P 0 05 P 0 01 Fig 2A and B 3 2 Immunofl uorescence histochemistry 3 2 1 TSG attenuates the loss of nigral TH positive neurons induced by MPTP The protective effects of TSG on the DAergic neurons in the SN of mice treated with MPTP are shown in Fig 3 There were sig nifi cant reductions in the number of TH positive cells in the SNpc for the MPTP group compared with the control group 34 2 2 77 P 0 01 In mice receiving daily oral administration of 40 mg kg TSG the number of TH positive cells was signifi cantly higher than those in MPTP vehicle treated mice 67 3 2 79 P 0 01 and it was even close to those of the control group 72 8 2 97 A simi lar but weaker effect was also seen in the 20 mg kg TSG treatment group 45 8 2 06 P 0 05 Moreover mice treated only with TSG did not demonstrate any signifi cant change in the number of SNpc TH positive cells compared to the controls Fig 3 3 3 Western blot analyses 3 3 1 TSG prevented the striatal DAT protein decrease induced by MPTP DAT play an important role in mediating the return of striatal dopamine Jakowec et al 2004 Western blots were performed on proteins from isolated striatal tissues to investigate the modulation of DAT protein in MPTP and or TSG treated mice Fig 4A MPTP treated mice had 40 lower striatal DAT protein levels compared with the control group P 0 05 How ever High dose TSG 40 mg kg treatments prevented this decrease P 0 01 Fig 4B 3 3 2 TSG prevented the striatal phosphor Akt Ser473 protein decrease induced by MPTP Akt is a pro survival kinase and is activated by the phosphor ylation at Ser473 via the PI3K pathway which is thought to be one of the molecules that promotes cell survival and prevents apo ptosis Franke et al 2003 Amaravadi and Thompson 2005 We investigated whether TSG mediated neuroprotection in PD mice implicated an Akt dependent pathway Fig 5A The phosphory lated forms at serine residue 473 of Akt pAkt were measured in these groups relative to their unphosphorylated form Striatal L Zhang et al Toxicology Letters 222 2013 155 163159 Fig 5 Western blot analysis protein level of p Akt Akt in the striatum of mice A Representative protein bands of p Akt and total Akt B The p Akt Akt ratio of relative protein level in MPTP NS group decreased obviously compared with the NS NS control group which could be prevented by the TSG signifi cantly Data are expressed as means SEM n 10 per group P 0 01 vs NS NS group P 0 05 P 0 01 vs MPTP NS group Fig 6 Western blot analysis protein level of p GSK3 GSK3 in the striatum of mice A Representative protein bands of p GSK3 and total GSK3 B The p GSK3 GSK3 ratio of relative protein level in MPTP NS group decreased obviously compared with the NS NS control group which could be prevented by the high dose of TSG signifi cantly Data are expressed as means SEM n 10 per group P 0 05 P 0 01 vs NS NS group P 0 05 P 0 01 vs MPTP NS group 160L Zhang et al Toxicology Letters 222 2013 155 163 Fig 7 Western blot analysis showing the ratio of antiapoptotic Bcl 2 on proapoptotic BAD protein level