This study observed the effects of ethanol extract of Gastrodiae Rhizoma(GE) on multiple aspects of mitochondrial dysfunction by investigating the mitochondria isolated from rat brains subjected to focal middle cerebral artery occlusion/reperfusion(MCAO/R). SD rats were randomly divided into a sham operation group(Sham), a model group(MCAO/R), low-and high-dose ethanol extract of GE groups(262.3 and 524.6 mg·kg~(-1)), and a nimodipine group(Nim, 15 mg·kg~(-1)). After continuous intragastric administration for 7 days, the MCAO/R model was induced in rats by the suture method. The neurological function and percentage of cerebral infarction volume were measured 24 h after reperfusion, and mitochondrial ultrastructure was observed under an electron microscope. Mitochondria were separated by gradient centrifugation and detected for reactive oxygen species(ROS), malondialdehyde(MDA), respiratory chain enzyme complex Ⅰ-Ⅳ activity, mitochondrial permeability transition pore(mPTP), mitochondrial membrane potential(MMP), and mitochondrial adenosine triphosphate(ATP) content. Enzyme-linked immunosorbent assay(ELISA) was used to detect the expression of cytochrome C(Cyt C) in different sites. TUNEL staining was used to observe the apoptosis of brain tissues in each group, and Western blot was used to detect the expression of B-cell lymphoma 2(Bcl-2) and Bcl-2-associated X protein(Bax) in brain tissues. The experimental results revealed that compared with the Sham group, the MCAO/R group showed evident neurological dysfunction and cerebral infarction(P<0.01) accompanied by mitochondrial swelling and crest disappearance, increased ROS level and MDA content, inhibited activity of respiratory chain enzyme complex Ⅰ-Ⅳ, abnormal opening of mPTP, and reduced MMP and mitochondrial ATP(P<0.01). Besides, many Cyt C was released from mitochondria into the cytoplasm to induce apoptosis(P<0.01). The ethanol extract of GE positively affected the behavior deficit and mitochondrial health of MCAO/R rats, with the manifestations of decreased production of ROS and MDA(P<0.01), potentiated activity of mitochondrial respiratory chain enzyme complex Ⅰ-Ⅳ, and restored the level of mPTP(P<0.05). In addition, the ethanol extract of GE reduced the loss of MMP and the escape of Cyt C to the cytoplasm(P<0.05), reduced the number of TUNEL positive cells(P<0.01) accompanied by the decrease in Bax and the up-regulation of Bcl-2(P<0.01), and increased the level of ATP(P<0.01). In conclusion, GE ethanol extract has a protective effect against MCAO/R-induced mitochondrial dysfunction, and the mechanism may be related to the regulation of oxidative stress, maintenance of functional morphology of mitochondria, and inhibition of endogenous apoptosis.
Animals ; Rats ; bcl-2-Associated X Protein/metabolism* ; Reactive Oxygen Species/metabolism* ; Ethanol ; 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology* ; Neuroprotective Agents/pharmacology* ; Rats, Sprague-Dawley ; Reperfusion Injury/metabolism* ; Infarction, Middle Cerebral Artery ; Mitochondria ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Apoptosis ; Cytochromes c/metabolism* ; Adenosine Triphosphate/pharmacology*

Wuzi-Yanzong-Wan (WZYZW) is a classic prescription for male infertility. Our previous investigation has demonstrated that it can inhibit sperm apoptosis via affecting mitochondria, but the underlying mechanisms are unclear. The purpose of the present study was to explore the actions of WZYZW on mitochondrial permeability transition pore (mPTP) in mouse spermatocyte cell line (GC-2 cells) opened by atractyloside (ATR). At first, WZYZW-medicated serum was prepared from rats following oral administration of WZYZW for 7 days. GC-2 cells were divided into control group, model group, positive group, as well as 5%, 10%, 15% WZYZW-medicated serum group. Cyclosporine A (CsA) was used as a positive control. 50 μmol·L^-1 ATR was added after drugs incubation. Cell viability was assessed using CCK-8. Apoptosis was detected using flow cytometry and TUNEL method. The opening of mPTP and mitochondrial membrane potential (MMP) were detected by Calcein AM and JC-1 fluorescent probe respectively. The mRNA and protein levels of voltage-dependent anion channel 1 (VDAC1), cyclophilin D (CypD), adenine nucleotide translocator (ANT), cytochrome C (Cyt C), caspase 3, 9 were detected by RT-PCR (real time quantity PCR) and Western blotting respectively. The results demonstrated that mPTP of GC-2 cells was opened after 24 hours of ATR treatment, resulting in decreased MMP and increased apoptosis. Pre-protection with WZYZ-medicated serum and CsA inhibited the opening of mPTP of GC-2 cells induced by ATR associated with increased MMP and decreased apoptosis. Moreover, the results of RT-qPCR and WB suggested that WZYZW-medicated serum could significantly reduce the mRNA and protein levels of VDAC1 and CypD, Caspase-3, 9 and CytC, as well as a increased ratio of Bcl/Bax. However, ANT was not significantly affected. Therefore, these findings indicated that WZYZW inhibited mitochondrial mediated apoptosis by attenuating the opening of mPTP in GC-2 cells. WZYZW-medicated serum inhibited the expressions of VDAC1 and CypD and increased the expression of Bcl-2, which affected the opening of mPTP and exerted protective and anti-apoptotic effects on GC-2 cell induced by ATR.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Animals ; Atractyloside/pharmacology* ; Cyclophilin D ; Male ; Matrix Metalloproteinases ; Mice ; Mitochondrial Membrane Transport Proteins/metabolism* ; Mitochondrial Permeability Transition Pore ; RNA, Messenger ; Rats

The aim of this study was to investigate the preventive and therapeutic effects of Erzhi Pills on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine( MPTP)-induced Parkinson's disease( PD) in mice,and explore its possible mechanism of action. Mice were intraperitoneally injected with MPTP( 30 mg·kg^-1,0. 01 m L·g^-1) once daily to induce PD for 8 days. In the treatment group,Erzhi Pills were given by intragastric administration( 2. 5 g·kg^-1,once daily for 30 days). The normal group received an equal volume of normalsaline. In terms of behavior,the limb movement coordination ability of the mice was detected by climbing,hanging and swimming experiments. The spatial learning and memory ability of the mice was detected by Morris water maze test. The content of MDA,as well as the activity of GSH-PX and SOD were determined in mice serum. Western blot was used to detect the protein expression levels of TH,MAOB and apoptosis-related factors CHOP and caspase-12 in brain tissues. Immunohistochemistry was used to detect the expression of TH in section of brain tissues in mice. The results showed that in behavioral aspects,as compared with the model group,the scores of limb movement ability as well as scores of spatial learning and memory ability were significantly improved in the treatment groups( P<0. 05). In terms of serological indicators,as compared with the model group,the activities of SOD and GSH-PX were significantly increased in the serum of treatment groups,and the content of MDA was significantly decreased( P<0. 05). The results of Western blot showed that as compared with the model group,the protein levels of TH in the brain tissues of the mice in treatment group were significantly up-regulated,while the protein levels of MAOB and apoptosis-related factors CHOP and caspase-12 were significantly down-regulated( P<0. 05). The results of immunohistochemistry showed that the number of TH positive cells in the brain tissues of the mice in the treatment group was significantly increased as compared with the model group( P<0. 05). In summary,Erzhi Pills have a certain preventive and therapeutic effect on MPTP-induced PD mice,which can significantly improve the limb motor coordination ability and spatial learning and memory ability of PD mice. Its mechanism may be related to down-regulating the expression of apoptosis-related factors CHOP and caspase-12,reducing the dopaminergic neuron damage and inhibiting dopaminergic neuronal apoptosis.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Animals ; Disease Models, Animal ; Drugs, Chinese Herbal/pharmacology* ; Mice ; Mice, Inbred C57BL ; Neuroprotective Agents/pharmacology* ; Parkinson Disease ; Substantia Nigra

OBJECTIVETo observe the effect of Baichanting Compound (BC) on dopamine (DA) in striatum of Parkinson's disease (PD) mice, and to screen the optimal component proportion.

METHODSThe PD model was established in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced C57BL/6 mice. By using uniform design, they were intervened by three extracts of BC in different proportions [Acanthopanax senticosus extract (X1): white peony root extract (X2): Uncaria rhynchophylla extract (X3) = 30.00: 34.92: 82.50, 48.00: 19.98: 72.19, 18.00: 44.88: 61.88, 36.00: 29.94: 51.56, 54.00: 15.00: 41.25, 24.00: 39.90: 30.94, 42.00: 24.96: 20.63). Equal volume of 5% carboxymethylcellulose sodium was administered to mice in the model group and the normal group by gastrogavage. All medication was lasted for 20 successive days. The dopamine (DA) content was determined by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS). Except 10 in the normal group, 20 PD model mice were screened and divided into the model group and the BC group (with the optimal proportion) according to random digit table. BC extract in optimal proportion was administered to mice in the BC group by gastrogavage, while equal volume of 5% carboxymethylcellulose sodium was administered to mice in the model group and the normal group by gastrogavage. All medication was lasted for 20 successive days. Praxiology was observed in each group. DA content in striatum was also detected. Results Compared with the normal group, the DA content in striatum decreased significantly in the model group (P < 0.01), suggesting a successful PD modeling. Compared with the model group, the DA content in striatum increased significantly in 1 and 2 groups (P<0.05). According to results of quadratic polynomial stepwise regression statistics, the regression equation obtained was: Y = 0.265 + 0.026 X 2 - 0.056 X 3 + 0.334 x 10(-3) x X1 x X3 + 0.691 x 10(-3) X X3(2). X3 extract was the main factor influencing the effectiveness (P < 0.01). The optimal proportion of BC was predicted by the regression equation: X1 = 54.00 mg/(kg x d), X2 = 44.88 mg/(kg x d), the X3 = 82.50 mg/(kg x d). The pole climbing time was shortened, times of autonomic activities increased, DA content was elevated, all with statistical difference in BC groups (P < 0.01, P < 0.05).

CONCLUSIONBC could increase DA content in PD model mice with the optimal proportion as 54.00: 44.88: 82.50.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Animals ; Disease Models, Animal ; Dopamine ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Mass Spectrometry ; Mice ; Mice, Inbred C57BL ; Motor Activity ; Parkinson Disease ; drug therapy ; metabolism

The aim of this study is to investigate the protective effect of N-[2-(4-hydroxyphenyl)ethyl]-2-(2, 5-dimethoxyphenyl)-3-(3-methoxy-4-hydroxyphenyl)acrylamide (FLZ), a novel synthetic squamosamide cyclic derivative, against Parkinson's disease (PD) model mice induced by the inflammatory bacterial endotoxin, lipopolysaccharides (LPS) and the neurotoxin 1-methy-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). C57/BL mice were ip injected LPS (5 mg x kg(-1)) once. One week following the LPS injection, mice received a subcutaneous injection of MPTP (25 mg x kg(-1)) once daily for 2 days. Eight weeks later, FLZ (25, 50 and 75 mg x kg(-1)) was orally administered to mice once daily for 60 days. The motor ability of the mice was evaluated by rod climbing test and footprint test. The dopamine (DA) levels in mouse striatum were determined by high performance liquid chromatography system. The tyrosine hydroxylase (TH)-positive cells were showed by immunohistochemical analysis. FLZ treatment significantly improved motor dysfunction of mice challenged by LPS plus MPTP. The increase of TH-positive cell numbers and elevation of DA levels may be contributed to the beneficial effects of FLZ on motor behavior. This study showed FLZ has significant therapeutic effect on LPS plus MPTP induced chronic PD model, which indicates its potential as a new candidate drug to treat PD.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; 3,4-Dihydroxyphenylacetic Acid ; metabolism ; Acrylamides ; pharmacology ; Animals ; Caffeic Acids ; pharmacology ; Corpus Striatum ; metabolism ; Dopamine ; metabolism ; Homovanillic Acid ; metabolism ; Lipopolysaccharides ; Male ; Mice ; Mice, Inbred C57BL ; Motor Activity ; drug effects ; Neurons ; drug effects ; metabolism ; Parkinson Disease, Secondary ; chemically induced ; metabolism ; pathology ; physiopathology ; Random Allocation ; Tyrosine 3-Monooxygenase ; metabolism

OBJECTIVETo investigate the effect of phosphorylated-ERK1/2 (p-ERK1/2) on inducible nitric oxide synthase (iNOS) expression in the substantia nigra (SN) of a mouse model of Parkinson's disease (PD), and explore the possible mechanism of dopaminergic (DA) neuron loss in the SN of the midbrain in PD.

METHODSPD was induced by intraperitoneal injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP) in C57BL/6N mice, and the behavioral changes of the PD mouse model were observed. Immunohistochemistry and Western blotting were used to detect the number of positive cells and the expressions of tyrosine hydroxylase (TH), p-ERK1/2 and iNOS in the SN of the PD mice, and their changes following Rg1 treatment were assessed.

RESULTSThe PD mice exhibited typical symptoms of PD, in which the number of TH-positive neurons and TH expression were significantly reduced by about 77% and 75% (P<0.01), respectively, 7 days after the 5th injection of MPTP as compared with those in the control group. Rg1 pretreatment significantly decreased the number of TH-positive neurons and TH expression by 44% and 41% (P<0.01), respectively. p-ERK1/2 expression was not observed in the cell nuclei until 1.5 h after the third injection of MPTP, and increased markedly at 6 h. Rg1 pretreatment significantly inhibited the expression of p-ERK1/2 and iNOS (P<0.01). A significant positive correlation was noted between the expression of p-ERK1/2 and iNOS (P<0.01).

CONCLUSIONP-ERK1/2 may regulate the expression of iNOS to induce DA neuron loss in the SN of PD, and Rg1 may protect the DA neurons possibly by depressing nuclear translocation of P-ERK1/2.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Animals ; Extracellular Signal-Regulated MAP Kinases ; chemistry ; pharmacology ; Ginsenosides ; pharmacology ; Male ; Mice ; Mice, Inbred C57BL ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Parkinson Disease, Secondary ; chemically induced ; enzymology ; Phosphorylation ; Substantia Nigra ; enzymology

OBJECTIVETo investigate the protective effects of cistanche total glycosides (CTG) on dopaminergic neuron in substantia nigra (SN) of model mice of Parkinson's disease (PD).

METHODSExperimental mice were randomly divided into 5 groups, the normal control group, the model group, the high (400 mg/kg), moderate (200 mg/kg) and low (100 mg/kg) dose CTG groups. Mouse model of chronic PD was induced by peritoneal injection of MPTP (1-methyl-4-phenyl-1,2,3,6-ttrahydropyridine) 30 mg/kg for 5 successive days. Climbing test was used to estimate the neurobehavior of mice on the 7th and 14th day (D7 and D14) after initiating MPTP injection; meantime, quantitative immunohistochemistry was conducted to detect the number of dopaminergic neuron in SN and expression of tyrosine hydroxylase (TH) in striatum.

RESULTSThe average time of climbing in the high dose CTG group on D7 and D14 was significantly shorter than that in the model group (P < 0.01). The mean optic density (OD) of TH in striatum was higher in the three CTG groups than that in the model group on D7 (P < 0.01); but on D14, significance only showed in the high and moderate dose CTG groups (P < 0.01). Moreover, the MPTP induced decrease of TH positive neuron could be antagonized by CTG, but significant difference only showed between the high dose CTG group and the model group at the two time points of observation (P < 0.05).

CONCLUSIONCTG could improve the neurobehavior of PD model mice significantly, and inhibit the decrease of nigral dopaminergic neurons and TH expression in striatum.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Animals ; Behavior, Animal ; drug effects ; Cistanche ; chemistry ; Dopamine ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Glycosides ; pharmacology ; Immunohistochemistry ; Male ; Mice ; Mice, Inbred C57BL ; Neurons ; drug effects ; metabolism ; pathology ; Neuroprotective Agents ; pharmacology ; Parkinson Disease, Secondary ; chemically induced ; physiopathology ; Random Allocation ; Substantia Nigra ; drug effects ; metabolism ; pathology ; Tyrosine 3-Monooxygenase ; metabolism

OBJECTIVETo investigate the inhibitory effect of antisense oligonucleotides (ODN) on dopamine transporter (DAT) in rats and observe the response of the rats to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

METHODSThe cannula was implanted in the substantia nigra compacta under a rat stereotaxic device, through which drugs were used. The rats with successful operation were divided randomly into four groups, and received injection of antisense, sense, missense oligonucleotides and saline respectively, in the substantia nigra compacta of each rat via the cannula, followed by MPTP (30 mg/kg) injection. Behavior of the rats was observed and immunohistochemistry was carried out to check the expression of DAT and apoptosis of dopamine cell.

RESULTSThe expression of DAT (positive unit, PU) in the substantia nigra compacta in rats was 6.65+/- 1.67 in the antisense ODN group, 12.41+/- 2.46 in saline group, 11.45+/- 1.17 in sense ODN group, and 10.35+/- 2.89 in missense ODN group. The expression of DAT was lower in the antisense ODN group than that of the other three groups (P< 0.01). The rotation of the rats induced by apomorphine was slower than that of the other three groups(P< 0.05). The apoptotic cells (21.4+/- 5.6) in the antisense ODN group (200x ) were less than that of the other three groups (61.6+/- 19.7, 56.5+/- 16.3, 52.2+/- 12.5 respectively), (P< 0.01).

CONCLUSIONThe expression of DAT can be inhibited effectively by the antisense ODN, and the response of the rats to the MPTP was reduced upon DAT inhibition.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; pharmacology ; Animals ; Apomorphine ; pharmacology ; Dopamine Plasma Membrane Transport Proteins ; genetics ; physiology ; Immunohistochemistry ; Male ; Oligonucleotides, Antisense ; genetics ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the role of P38 signaling pathway in modulating the expression of cyclooxygenase-2 (COX-2) in the substantia nigra (SN) of mice with 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced Parkinson disease (PD), and explore the possible mechanism of the dopaminergic (DA) neuron death in PD and the effects of ginsenoside Rg1 on the P38 signaling pathway and DA neurons.

METHODSC57BL6 mice were treated with MPTP to produce the subacute PD model, and the behavioral changes were observed. Immunohistochemistry and Western blotting for tyrosine hydroxylase (TH), COX-2, prostaglandin E2 (PGE2) and phosphorylated P38 (p-P38) were used to observe the changes of positive cell number in the midbrain after treatment with ginsenoside Rg1.

RESULTSCompared with the control mice, the mice with PD presented with typical symptoms of PD. The number of p-P38-, COX-2-, and PGE2-positive cells significantly increased in the SN area 6 h after the 3rd injection of 30 mg/kg MPTP (P<0.01). The number of TH-positive neurons in the PD model group was substantially reduced by about 60% (P<0.01) in 24 h after the 5th injection of MPTP. In mice with ginsenoside Rg1 treatment, the number of p-P38-, COX-2-, and PGE2-positive cells was reduced obviously 6 h after the 3rd injection of MPTP as compared with that in the model group (P<0.01). The number of TH-positive neurons in the SN was decreased by only 30% (P<0.01 vs control group) 24h after the 5th injection of MPTP.

CONCLUSIONP38 signaling pathway may play an important role in modulating COX-2 expression in the SN in the early stage of MPTP-induced subacute PD, and ginsenoside Rg1 may act on the P38 signaling pathway to protect the DA neurons in PD.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Animals ; Blotting, Western ; Cyclooxygenase 2 ; biosynthesis ; Ginsenosides ; pharmacology ; Immunohistochemistry ; Male ; Mice ; Mice, Inbred C57BL ; Neurons ; drug effects ; metabolism ; pathology ; Parkinson Disease, Secondary ; chemically induced ; metabolism ; physiopathology ; Signal Transduction ; drug effects ; Substantia Nigra ; drug effects ; metabolism ; pathology ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo investigate the effect of phophorylated c-Jun (p-c-Jun) expression on the expression of COX-2 in the substantia nigra (SN) of the MPTP mouse model of subacute Parkinson disease (PD) and explore the possible mechanism of the dopaminergic (DA) neuron death in PD.

METHODSC57BL/6N mice were treated with MPTP to establish subacute PD model. The changes of TH-, COX-2- and p-c-Jun-positive cells, and the expression levels of TH, COX-2 and p-c-Jun in the SN in the midbrain were observed with inmmunohistochemistry and Western blotting before and after administration of SP600125, a specific JNK inhibitor.

RESULTSCompared with the mice in control group, the PD mice exhibited typical symptoms of PD. The number of TH-positive neurons and expression level of TH in the model group were significantly reduced in the substantia nigra by about 65% and 75% (P<0.001) 7 days after the fifth injection of MPTP. The number of COX-2-immunoreactive cells and the expression level of COX-2 were significantly increased. P-c-Jun was specifically expressed in the nuclei of neurons and p-c-Jun expression level was significantly increased in the SN 6 h after the third injection of MPTP. Double-labeling immunofluorescence assay showed coexpression of COX-2 and p-c-Jun in TH-positive neurons in the SN. In mice treated with JNK inhibitor, the number of TH-positive neurons and TH expression level in the SN was only decreased by 15% and 20% as compared with the control group (P<0.001) 7 days after the fifth injection of MPTP, COX-2-positive cell number and COX-2 expression level were obviously reduced as compared with the model group (P<0.001), and p-c-Jun was expressed mainly in the cytoplasm of the neurons whose expression level in SN were significantly decreased 6 h after the third injection of MPTP. The PD mice treated with SP600125 showed slight behavioral symptoms.

CONCLUSIONP-c-Jun expression may play an important role in mediating COX-2 expression in the SN in the MPTP model of subacute PD, and inhibiting p-c-Jun activity may provide neuroprotection to the mouse model.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; pharmacology ; Animals ; Anthracenes ; pharmacology ; Blotting, Western ; Cell Death ; drug effects ; Cyclooxygenase 2 ; metabolism ; Disease Models, Animal ; Dopamine ; metabolism ; Gene Expression Regulation, Enzymologic ; drug effects ; Humans ; Immunohistochemistry ; MAP Kinase Signaling System ; drug effects ; Male ; Mice ; Mice, Inbred C57BL ; Neurons ; pathology ; Parkinson Disease ; enzymology ; etiology ; metabolism ; pathology ; Phosphoproteins ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-jun ; metabolism ; Substantia Nigra ; drug effects ; metabolism