Sleep-wake disorder is one of the most common nonmotor symptoms of Parkinson's disease (PD). Melatonin has the potential to improve sleep-wake disorder, but its mechanism of action is still unclear. Our data showed that melatonin only improved the motor and sleep-wake behavior of a zebrafish PD model when melatonin receptor 1 was present. Thus, we explored the underlying mechanisms by applying a rotenone model. After the PD zebrafish model was induced by 10 nmol/L rotenone, the motor and sleep-wake behavior were assessed. In situ hybridization and real-time quantitative PCR were used to detect the expression of melatonin receptors and lipid-metabolism-related genes. In the PD model, we found abnormal lipid metabolism, which was reversed by melatonin. This may be one of the main pathways for improving PD sleep-wake disorder.
Animals ; Zebrafish ; Melatonin/pharmacology* ; Lipid Metabolism/drug effects* ; Disease Models, Animal ; Rotenone/pharmacology* ; Sleep Wake Disorders/metabolism* ; Parkinson Disease/metabolism* ; Motor Activity/drug effects* ; Sleep/drug effects*

OBJECTIVEThe main objective of this study was to preliminarily determine the optimum formulation of a Chinese herbal formula that may have neuroprotective effects against rotenone-induced Parkinson's disease (PD).

METHODSSeven recipes were made from Dihuang (DH, Rehmannia glutinosa Libosch), Roucongrong (RCR, Cistanche deserticola Y.C.Ma), Niuxi (NX, Achyranthes bidentata Bl.) and Shanzhuyu (SZY, Cornus officinalis Sieb. et Zucc) in different proportions, according to the principles of uniform design (4 factors 7 levels). Tyrosine hydroxylase (TH)-positive neurons in substantia nigra pars compacta (SNpc) were detected by immunohistochemistry and rotenone-exposure days necessary to induce PD symptoms were recorded. To probe one likely mechanism of the formulas, echinacoside (ECH) concentrations of all seven recipes were determined by high-performance liquid chromatography and related to number of TH-positive neurons.

RESULTSThe data showed that recipe 4 (DH:RCR:SZY:NX = 1:1:1:1) and recipe 7 (DH:RCR:SZY:NX = 7:5:3:1) partially reversed rotenone-induced death of TH-positive neurons in the SNpc and significantly increased rotenone-exposed days compared with model group. Pharmacologically, there was not a strong correlation between ECH concentration and TH-positive neurons.

CONCLUSIONThe investigated formulations of Chinese herbs had neuroprotective effects against PD models, and the neuroprotective effects were weakly related to the proportion of key herbs. However the neuroprotective effects of the formula may not result from a single active constituent.

Animals ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; Humans ; Male ; Neuroprotective Agents ; administration & dosage ; chemistry ; Parkinson Disease ; drug therapy ; etiology ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar ; Rotenone ; adverse effects

PURPOSE: Rotenone is the most widely used neurotoxin for the making Parkinson disease (PD) animal model. The neurodegenerative disorder PD shows symptoms, such as slowness of movements, tremor at resting, rigidity, disturbance of gait, and instability of posture. We investigated whether treadmill running improves motor ability using rotenone-caused PD rats. The effect of treadmill running on PD was also assessed in relation with apoptosis of cerebellar Purkinje cells. METHODS: Treadmill running was applied to the rats in the exercise groups for 30 minutes once a day for 4 weeks, starting 4 weeks after birth. We used rota-rod test for the determination of motor coordination and balance. In this experiment, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, immunohistochemistry for calbindin, glial fibrillary acidic protein (GFAP), Iba-1, and western blot analysis for Bax and Bcl-2 were performed. RESULTS: Treadmill running enhanced motor balance and coordination by preventing the loss of Purkinje cells in the cerebellar vermis. Treadmill running suppressed PD-induced expression of GFAP-positive reactive astrocytes and Iba-1-positive microglia, showing that treadmill running suppressed reactive astrogliosis and microglia activation. Treadmill running suppressed TUNEL-positive cell number and Bax expression and enhanced Bcl-2 expression, demonstrating that treadmill running inhibited the progress of apoptosis in the cerebellum of rotenone-induced PD rats. CONCLUSIONS: Treadmill running improved motor ability of the rotenone-induced PD rats by inhibiting apoptosis in the cerebellum. Apoptosis suppressing effect of treadmill running on rotenone-induced PD was achieved via suppression of reactive astrocyte and inhibition of microglial activation.
Animals ; Apoptosis ; Astrocytes ; Blotting, Western ; Calbindins ; Cell Count ; Cerebellar Vermis ; Cerebellum ; Gait ; Glial Fibrillary Acidic Protein ; Immunohistochemistry ; Microglia ; Models, Animal ; Neurodegenerative Diseases ; Parkinson Disease* ; Parturition ; Posture ; Purkinje Cells* ; Rats* ; Rotenone ; Running ; Tremor

OBJECTIVES: The aim of this study was whether quercetin induces cell death by caspase and MAPK signaling pathway in EGFR mutant lung cancer cells. METHODS: PC-9 cells, EGFR mutant lung cancer cells, were treated various times and concentrations of quercetin and harvested and measured using MTT assay, DNA fragmentation, Western blotting, and FACS analysis. RESULTS: Treatment with quercetin in PC-9 cells resulted in inhibition of cell growth through apoptosis. Quercetin-induced apoptosis was associated with caspase-dependent manner. Quercetin also significantly increased levels of phosphor-p38 and decreased levels of phosphor-ERK, indicating that quercetin induces p38 MAPK signaling pathway in PC-9 cells. Quecetin treatment also generated the release of cytochrome c in PC-9 cells; however, pretreatment with rotenone or z-LEHD-fmk, significantly attenuated quercetin-induced apoptosis. CONCLUSIONS: Our data indicate that quercetin exhibits EGFR mutant lung cancer effects through apoptosis by caspase dependent and mitochondrial pathway.
Apoptosis ; Blotting, Western ; Cell Death* ; Cytochromes c ; DNA Fragmentation ; Lung Neoplasms* ; Lung* ; Mitochondria ; p38 Mitogen-Activated Protein Kinases* ; Quercetin* ; Rotenone

Cryptotanshinone (CPT) is a natural compound isolated from traditional Chinese medicine Salvia miltiorrhiza Bunge. In the present study, the regulatory effect and potential mechanisms of CPT on tumor necrosis factor alpha (TNF-α) induced lectin-like receptor for oxidized low density lipoprotein (LOX-1) were investigated. Human umbilical vein endothelial cells (HUVECs) were cultured and the effect of TNF-α on LOX-1 expression at mRNA and protein levels was determined by Real-time PCR and Western blotting respectively. The formation of intracellular ROS was determined with fluorescence probe CM-DCFH2-DA. The endothelial ox-LDL uptake was evaluated with DiI-ox-LDL. The effect of CPT on LOX-1 expression was also evaluated with SD rats. TNF-α induced LOX-1 expression in a dose- and time-dependent manner in endothelial cells. TNF-α induced ROS formation, phosphorylation of NF-κB p65 and ERK, and LOX-1 expression, which were suppressed by rotenone, DPI, NAC, and CPT. NF-κB inhibitor BAY11-7082 and ERK inhibitor PD98059 inhibited TNF-α-induced LOX-1 expression. CPT and NAC suppressed TNF-α-induced LOX-1 expression and phosphorylation of NF-κB p65 and ERK in rat aorta. These data suggested that TNF-α induced LOX-1 expression via ROS activated NF-κB/ERK pathway, which could be inhibited by CPT. This study provides new insights for the anti-atherosclerotic effect of CPT.
Animals ; Aorta ; Blotting, Western ; Endothelial Cells* ; Fluorescence ; Human Umbilical Vein Endothelial Cells ; Lipoproteins ; Medicine, Chinese Traditional ; Phosphorylation ; Rats ; Reactive Oxygen Species* ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; Rotenone ; Salvia miltiorrhiza ; Tumor Necrosis Factor-alpha

OBJECTIVE: To investigate the effect of the L10P mutation on the cellular mitochondrial disfunction. METHODS: Spectrophotometer, flow cytometry and electron microscope was utilized to examine cell viability, reactive oxygen species (ROS), mitochondrial transmembrane potential, complex I activity and mitochondrial morphous of the HEK293 monoclone cell lines, in which wild-type and L10P mutant DJ-1 protein are stably expressed. RESULTS: Compared with the cell lines expressing empty vector, we found the ROS levels were elevated, the cell viability, mitochondrial transmembrane potential, complex I activity were reduced in the cells expressing L10P mutant DJ-1 protein (P<0.05). We also found mitochondria in these cells were swelling and some mitochondria were vacuolar degeneration. These phenomena were more obvious when rotenone was used. But in the cells expressing wild-type DJ-1, ROS levels were lower, the cell viability, mitochondrial transmembrane potential, and complex I activity were higher than other cell lines (P<0.05), especially under the induction of rotenone. These results suggested that L10P mutant DJ-1 protein probably lost the ability of anti-oxidative stress and affect the normal function of mitochondria. CONCLUSION The L10P DJ-1 mutation results in a toxic protein, which lacks the protective function of wild-type protein on mitochondria due to the decrease in the ability of anti-oxidative stress.
Cell Survival ; HEK293 Cells ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; Membrane Potential, Mitochondrial ; Mitochondria ; pathology ; Mutation ; Oncogene Proteins ; genetics ; Oxidative Stress ; Protein Deglycase DJ-1 ; Reactive Oxygen Species ; metabolism ; Rotenone

OBJECTIVETo examine the effect of a selective inhibitor of 5-lipoxygenase (5-LOX) zileuton on microglia-mediated rotenone neurotoxicity.

METHODSThe supernatant from different concentrations of rotenone-stimulated mouse microglia BV2 cells was used as the conditioned media (CM) for PC12 cells. The viability of PC12 cells was determined by MTT assay and lactate dehydrogenase (LDH) release. Cell death was observed by LDH release and double fluorescence staining with Hoechst/propidiumiodide (PI). The effect of zileuton on microglia-mediated rotenone toxicity was evaluated by the above methods.

RESULTSRotenone at 1-10 nmol/L was nontoxic to PC12 cells directly. However, the CM from BV2 cells that were treated with rotenone (1-10 nmol/L) resulted in toxicity of PC12 cells. The BV2 CM which stimulated with rotenone (1-10 nmol/L) induced morphological changes, reduced cell viability, and increased LDH release and cell necrosis in PC12 cells. Pretreatment of BV2 cells with the 5-LOX inhibitor zileuton (0.01-1 μmol/L) protected PC12 cells from the microglia-mediated rotenone toxicity.

CONCLUSIONThe 5-LOX inhibitor zileuton effectively attenuates microglia-mediated rotenone toxicity in PC12 cells. These results suggest that 5-LOX pathway may be involved in neuronal death induced by microglial inflammation.

Animals ; Cell Death ; drug effects ; Cells, Cultured ; Hydroxyurea ; analogs & derivatives ; pharmacology ; Lipoxygenase Inhibitors ; pharmacology ; Mice ; Microglia ; cytology ; PC12 Cells ; Rats ; Rotenone ; toxicity

OBJECTIVETo explore the protective effect of baicalin against rotenone-induced injury on PC12 cells, and the po-tential mechanism of action action was also explored.

METHODPC12 cells were injured by rotenone and were treated with different concentrations (0.1, 1, 10 μmol x L(-1)) of baicalin at the same time. Cell viability was analyzed by MTT, and morphology was observed by phase-contrast microscopy. The cell apoptosis was detected by flow cytometry by Annexin V-FITC/PI staining. The intracellular ROS level was determined by fluorescence microscope with DCF-DA staining. The expression of Bcl-2, Bax and Caspase-3 was analyzed by Western blot.

RESULTThe viability of PC12 cells exposure to rotenone for 24 hour was gradually decreased with dose escalating and 1.5 μmol x L was adopted to do the following experiment. Baicalin increased cell viability, improved cell morphology and decreased intracellular ROS level. Moreover, FACS indicated baicalin attenuated the apoptosis induced by rotenone significantly. Western blot showed that Bcl-2, Bax and Caspase-3 expression in rotenone-induced PC12 cells was reversed by baicalin.

CONCLUSIONThis study has demonstrated that baicalin protects PC12 cells against rotenone-induced apoptosis, at least in part, by scavenging excessive ROS and inhibiting the mitochondrion-dependent apoptotic pathway.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Survival ; drug effects ; Cytoprotection ; drug effects ; Flavonoids ; pharmacology ; Gene Expression Regulation ; drug effects ; Intracellular Space ; drug effects ; metabolism ; PC12 Cells ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Reactive Oxygen Species ; metabolism ; Rotenone ; pharmacology ; bcl-2-Associated X Protein ; metabolism

Rotenone is one of the typical inhibitors of the complex I on the mitochondrial respiratory chain. Numerous studies showed when applied to live animals or cells, rotenone could lead to mitochondrial dysfunction, ROS augment, and thus oxidative damage to proteins, lipids and nucleic acids. Through exploring the process of ROS generation in mitochondria, the relationship between rotenone and mitochondrial ROS generation and the role of rotenone in DNA damage, we elucidated the mechanisms of rotenone induced-mitochondrial oxidative damage. At the same time, we attempted to explore the mtDNA damage and the mutation induced by rotenone.
Animals ; DNA Damage ; DNA, Mitochondrial ; Mitochondria ; pathology ; Mutation ; Oxidative Stress ; Reactive Oxygen Species ; metabolism ; Rotenone ; metabolism

The underlying mechanism of deguelin regulating the cell cycle in human Burkitt's lymphoma cell line Raji cells in vitro, and the cytotoxicity of deguelin to Raji cells and human peripheral blood monocular cells (PBMCs) were investigated. The effects of deguelin on the growth of Raji cells were studied by 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Apoptosis was detected through Hoechst 33258 staining. The effect of deguelin on the cell cycle of Raji cells was studied by a propidium iodide method. The expression levels of cyclin D1, P21 and pRb were examined by using Western blotting. The results showed that the proliferation of Raji cells was inhibited in the deguelin-treated group, with a 24-h IC(50) value of 21.61 nmol/L and a 36-h IC(50) value of 17.07 nmol/L. Proliferation in Raji cells was inhibited significantly by deguelin, while little change was observed in PBMCs. Deguelin induced G(2)/M arrest in Raji cells. The expression of cyclin D1, P21 and pRb was dramatically down-regulated by deguelin in a dose-dependent manner. It was concluded that deguelin could inhibit the proliferation of Raji cells by arresting the cells at G(2)/M phase and inducing the cell apoptosis. Moreover, deguelin selectively induced apoptosis of Raji cells with low toxicity to PBMCs. The antitumor effects of deguelin were related to the down-regulated expression of cyclin D1, P21 and pRb proteins.
Cell Cycle ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Humans ; Rotenone ; analogs & derivatives ; pharmacology