C-phycocyanin shows neuroprotective effect against rotenone-induced Parkinson’s disease in mice

Objective:To investigate the neuroprotective effect of C-phycocyanin in a mouse model of rotenone-induced Parkinson’s disease.Methods:C-phycocyanin(50 mg/kg,i.p.,daily)was administered to rotenone(30 mg/kg,p.o.,daily)treated mice for 28 days.Behavioral studies(Y-maze,rotarod,round beam walk,and wire-hang tests)were carried out to assess neurobehavioral deficits.Glutathione and malondialdehyde were determined in both serum and striatal tissue.Molecular proteins(AKT,AMPK,NF-κB,BDNF,and alpha-synuclein)in the striatum were estimated using ELISA.Histopathological analyses(hematoxylin and eosin stainning as well as Nissl staining)were carried out to assess structural abnormalities in the striatum.Results:C-phycocyanin significantly increased BDNF levels and decreased alpha-synuclein levels.It also slightly upregulated AMPK and AKT levels without significant difference compared with the rotenone group.Additionally,rotenone-induced elevated oxidative stress and structural abnormalities in the striatum were markedly mitigated by C-phycocyanin.Conclusions:C-phycocyanin might have potential neuroprotective effects against Parkinson’s disease.Further studies are warranted to verify its efficacy and to understand the molecular mechanisms behind the neuroprotective effects of C-phycocyanin in Parkinson’s disease.

α-Synuclein redistributed and aggregated in rotenone-induced Parkinson’s disease rats

Objective To observe the influence of rotenone on the distribution of α-synuclein （ASN） in rat model of Parkinson＇s disease （PD）. Methods Wistar rats were randomly divided into two groups and received 2 mg/kg rotenone （s.c.） or sunflower oil （as control group） for about 4 weeks. The hippocampus, substantia nigra and striatum of brain were observed. Hematoxylin and eosin stain were used to observe the Lewy body like inclusion. The expression of tyrosine hydroxylase （TH） or ASN protein was determined by anti-TH or anti-α-synuclein immunohistochemistry, respectively. Results In control rats, ASN protein distributed widely in brain, especially in hippocampus, cortex and striatum. Rotenone obviously increased TH positive neurons and fibers loss in substantia nigra and striatum （P 〈 0.05）. In rotenone treated rats, ASN positive cells increased in global brain but not distributed in an even manner. In substantia nigra, ASN positive stuff was found aggregate in both cytoplasm and nucleus, and some formed spherical inclusion; in striatum, ASN positive neurites end aggregated and agglomerated around neurons; and in hippocampus, few dot-like ASN were aggregated in cell body, and no notable change was found in nucleus. Conclusion In rotenone administrated PD rats, ASN protein aggregated in several brain regions but most obviously in striatum and substantia nigra, and the distribution region of ASN was changed from peri-synapse to the cytoplasm and nucleus of dopaminergic neuron.

Synthesis, Crystal Structure and Biological Activites of Rotenone O-Alkyl Oximes

A series of rotenone O-alkyl oxime derivatives was designed and synthesized. Their structures were confirmed by elemental analyses, Fourier transform infrared（FTIR） and 1H NMR spectral studies, and the typical crystal structure of rotenone O-ethyl oxime（3b） was determined by X-ray diffraction. The preliminary biological activities of the new compounds were evaluated. The results of bioassays indicate that the title compounds exhibit moderate insecticidal and bactericidal activities. Among the synthesized compounds, compound 3q exhibited 90.0% mortality against M. separata at 1000 μg/mL. Compounds 3b and 3g exhibited both 90.0% inhibition rate against R. solani at 500 μg/mL, respectively.

Gastrodin inhibits neuroinflammation in rotenone-induced Parkinson's disease model rats

The present study showed that the latency of rats moving on a vertical grid was significantly prolonged, and the number of rats sliding down from the declined plane was increased remarkably, in rotenone-induced Parkinson＇s disease model rats compared with control rats. The moving latency recovered to normal levels, but the number of slides was significantly increased at 28 days after model establishment. The slope test is a meaningful approach to evaluate the symptoms of Parkinson＇s disease model rats treated with rotenone. In addition, loss of substantia nigral dopaminergic neurons in model rats was observed at 1 day after the model was established, and continued gradually at 14 and 28 days. The expression of tyrosine hydroxylase-positive cells was significantly increased in gastrodin-treated rats at 14 days. Significant numbers of activated microglia cells were observed in model rats at 14 and 28 days; treatment of rats with Madopar at 28 days suppressed microglial activation. Treatment of rats with gastrodin or Madopar at 28 days significantly reduced interleukin-1β expression. The loss of substantia nigral dopaminergic neurons paralleled the microglial activation in Parkinson＇s disease model rats treated with rotenone. The inflammatory factors tumor necrosis factor-a and interleukin-1β are involved in the substantia nigral damage. Gastrodin could protect dopaminergic neurons via inhibition of interteukin-1β expression and neuroinflammation in the substantia nigra.

Effect of moxibustion on mTOR-mediated autophagy in rotenone-induced Parkinson's disease model rats

Defects in autophagy-mediated clearance of α-synuclein may be one of the key factors leading to progressive loss of dopaminergic neurons in the substantia nigra. Moxibustion therapy for Parkinson’s disease has been shown to have a positive effect, but the underlying mechanism remains unknown. Based on this, we explored whether moxibustion could protect dopaminergic neurons by promoting autophagy mediated by mammalian target of rapamycin （mTOR）, with subsequent elimination of α-syn. A Parkinson’s disease model was induced in rats by subcutaneous injection of rotenone at the back of their necks, and they received moxibustion at Zusanli （ST36）, Guanyuan （CV4）and Fengfu （GV16）, for 10 minutes at every point, once per day, for 14 consecutive days. Model rats without any treatment were used as a sham control. Compared with the Parkinson’s disease group, the moxibustion group showed significantly greater tyrosine hydroxylase immunoreactivity and expression of light chain 3-II protein in the substantia nigra, and their behavioral score, α-synuclein immunoreactivity,the expression of phosphorylated mTOR and phosphorylated ribosomal protein S6 kinase （p-p70S6K） in the substantia nigra were significantly lower. These results suggest that moxibustion can promote the autophagic clearance of α-syn and improve behavioral performance in Parkinson’s disease model rats. The protective mechanism may be associated with suppression of the mTOR/p70S6K pathway.

H_2O_2预处理抑制α-Synuclein的聚集并减轻Rotenone对PC12细胞的损伤

目的探讨H2O2预处理(HPP)对Rotenone诱导PC12细胞损伤及α-Synuclein聚集的影响。方法采用四甲基偶氮唑盐法(MTT法)、流式细胞仪、免疫印迹(western blot)技术及共聚焦显微镜分别检测细胞活力、细胞凋亡率、α-Synuclein的表达及聚集。结果H2O2预处理能够减少α-Synuclein寡聚体的表达及聚集体的形成,增加PC12细胞活力(Rot组47%±3.67%,HPP+Rot组82%±6.69%),抑制细胞凋亡(Rot组49.68%±4.55%,HPP+Rot组18.73%±5.64%)。结论H2O2预处理能减轻Rotenone对PC12细胞的毒性损伤作用,这种保护作用可能是通过抑制α-Synuclein寡聚体表达实现的。

Neurotoxicity of the pesticide rotenone on neuronal polarization: a mechanistic approach

Neurons are the most extensive and polarized cells that display a unique single long axon and multiple dendrites, which are compartments exhibiting structural and functional differences. Polarity occurs early in neuronal development and it is maintained by complex subcellular mechanisms throughout cell life. A well-defined and controlled spatio-temporal program of cellular and molecular events strictly regulates the formation of the axon and dendrites from a non-polarized cell. This event is critical for an adequate neuronal wiring and therefore for the normal functioning of the nervous system. Neuronal polarity is very sensitive to the harmful effects of different factors present in the environment. In this regard, rotenone is a crystalline, colorless and odorless isoflavone used as insecticide, piscicide and broad spectrum pesticide commonly used earlier in agriculture. In the present review we will summarize the toxicity mechanism caused by this pesticide in different neuronal cell types, focusing on a particular biological mechanism whereby rotenone could impair neuronal polarization in cultured hippocampal neurons. Recent advances suggest that the inhibition of axonogenesis produced by rotenone could be related with its effect on microtubule dynamics, the actin cytoskeleton and their regulatory pathways, particularly affecting the small RhoGTPase RhoA. Unveiling the mechanism by which rotenone produces neurotoxicity will be instrumental to understand the cellular mechanisms involved in neurodegenerative diseases influenced by this environmental pollutant, which may lead to research focused on the design of new therapeutic strategies.

Parallel relationship between microglial activation and substantia nigra damage in a rotenone-induced Parkinson's disease rat model

BACKGROUND： Inflammatory injury induced by microglial activation plays an important role in the occurrence and development of Parkinson＇s disease （PD）. However, few studies have examined the relationship between microglia and substantia nigra damage or dopaminergic neuron loss in animals with rotenone-induced PD.OBJECTIVE： To explore the relationship between activated microglia and loss of the substantia nigra, and the changes in concentration and dose of rotenone in the brain of rats with rotenone-induced PD.DESIGN, TIME AND SETTING： The neuropathological experiment was performed at the School of Traditional Chinese Medicine, Capital Medical University, China, from July 2007 to July 2008. MATERIALS： Rotenone was purchased from Sigma, USA. METHODS： The Parkinson＇s model was induced by injection of a rotenone oily-emulsion （2 mg/kg daily） subcutaneously into the back of 58 male adult Wistar rats for 3-6 weeks. Another three rats served as normal controls.MAIN OUTCOME MEASURES： Neurobehavioral changes were observed and recorded following rotenone treatment. Tyrosine hydroxylase and complement receptor OX42 were separately analyzed by immunohistochemical staining within 4 weeks following stopping rotenone treatment. Rotenone content was measured using high performance liquid chromatography in the cerebellum of rats that scored 2.4-6.RESULTS： Rotenone induced a loss of dopaminergic neurons in the substantia nigra as well as microglial activation, with increased behavior scores. Dopaminergic loss was still ongoing even when rotenone was stopped. Dopaminergic neuronal degeneration in the substantia nigra was initially 6%, but was 85% at 2 weeks after scoring, and degeneration depended on activated microglia. Rotenone was detected in the cerebellum at concentrations between 78.9 μg/L and 309.6 μg/L. CONCLUSION： Nigrostriatal dopaminergic degeneration paralleled the microglial activation. Rotenone absorbed into the brain in its original form initiated pathological injury in the substantia niara of PD rats.

Determination of Rotenone Residues in Foodstuffs by Solid-Phase Extraction (SPE) and Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS)

We developed a novel approach to determine rotenone residues in foodstuffs, by integrating solid-phase extraction （SPE） and liquid chromatography/tandem mass spectrometry （LC-MS/MS） technologies, to achieve high sensitivity and selectivity. In our method, the solvent extraction with n-hexane-dichloromethane （50：50, v/v） and cleanup with florisil SPE cartridges using ethyl acetate-ethyl ether （25：75, v/v） as eluents provided adequate recovery of rotenone. The detection of rotenone was then carried out by LC-MS/MS using acetonitrile-water with the 0.1% formic acid （w/v） as the mobile phase. The multiple reaction monitoring （MRM） scheme employed in the approach involved the transitions of the precursor ion to three selected product ions, in which one pair for quantification was m/z 395.3 〉 213.2 and the other two pairs for identification were m/z 395.3 〉 192.2 and 395.3 〉 367.0. The limits of quantification （LOQs） of the method ranged from 0.001 to 0.005 mg kg-1 depending on the matrix. Intra- and inter-day precisions （relative standard deviations, RSDs） for rotenone were less than 7.1 and 14.8%, respectively. Results from repetitive analysis suggested good reproducibility of the method for rotenone residue detection. The recoveries at three concentrations （LOQ, 10LOQ and 100LOQ） ranged from 79.3-118.3% in cabbage, potato, onion, carrot, apple, orange, banana, lichee, tea, and Shiitake mushroom. The proposed procedure was then applied to the analysis of 129 real samples collected from Xiamen, Fujian Province, China. The existence of rotenone was found in two tea products with concentrations of 0.012 and 0.016 mg kg-1, respectively. The method has great potential for routine analysis of monitoring rotenone residue in foodstuffs.

Effect of rifampicin pre-and post-treatment on rotenone-induced dopaminergic neuronal apoptosis and alpha-synuclein expression

BACKGROUND： Rifampicin inhibits the formation of a-synuclein multimer and protects against 1-methyl-4-phenyl-1,2, 3, 6-tetrahydropyritine （MPTP）-induced PC12 cell apoptosis. OBJECTIVE： To compare the effect of rifampicin pre- and post-treatment on tyrosine hydroxylase and α-synuclein expression in substantia nigra pars compacta in a rat model of Parkinson＇s disease. DESIGN, TIME AND SE＇B＇ING： A randomized, controlled experiment was performed at the Experimental Animal Center of Sun Yat-sen University North Campus （China） from November 2006 to October 2008. MATERIALS： Rifampicin was purchased from MD, USA; rotenone was purchased from Sigma, USA; mouse anti-rat α-synuclein monoclonal antibody was purchased from B＆D, USA; and rabbit anti-rat tyrosine hydroxylase monoclonal antibody was purchased from Chemicon, USA. METHODS： A total of 72 male, Sprague Dawley rats, aged 8 weeks, were randomly assigned to 5 groups： blank control （n = 12）, rifampicin （n = 12）, rotenone （n = 16）, rifampicin pre-treatment （n = 16）, and rifampicin post-treatment （n = 16）. Parkinson＇s disease model rats were established via a subcutaneous injection of rotenone （1.5 mg/kg per day） in the three treatment groups, once a day for 3 successive weeks. Rifampicin （30 mg/kg per day） was intragastrically administered in the rifampicin pre-treatment group 3 days prior to rotenone induction and in the rifampicin post-treatment group 7 days after rotenone induction. Rats were treated with a subcutaneous injection of 1 mL/kg per day sunflower oil in the blank control group and an intragastric injection of 30 mg/kg per day rifampicin in the rifampicin group, once a day for 3 successive weeks in total. MAIN OUTCOME MEASURES： Prior to treatment and in the end of the 3^rd week after treatment, the rats were evaluated using the modified neurological severity score. The substantia nigra from the rats was extracted for hematoxylin-eosin staining. Western blot analysis was performed to determine tyrosine hydroxylase and α-synuclein expression. RESULTS： Hematoxylin-eosin staining revealed a significant reduction in the number of substantia nigral neurons in the rotenone group, in addition to neurodegradation, hypopigmentation, and pyknosis. In the rifampicin pre-treatment and post-treatment groups, the number of dopaminergic neurons was significantly increased compared with the rotenone group （P 〈 0.01）, with slight neuronal damage. Compared with the rotenone group, substantia nigral tyrosine hydroxylase expression was significantly increased in the rifampicin pre-treatment and post-treatment groups （P 〈 0.01）, but α-synuclein expression and modified neurological severity scores were significantly decreased （P 〈 0.01）. In addition, the effect of rifampicin in the pre-treatment group was superior to the post-treatment group. There was no significant difference in tyrosine hydroxylase and α-synuclein expression, or in the modified neurological severity scores, between the blank control and rifampicin groups （P 〉 0.05）. CONCLUSION： Rifampicin significantly attenuated neuropathological and behavioral motor deficits induced by rotenone. Moreover, rifampicin enhanced tyrosine hydroxylase expression, but inhibited α-synuclein expression. The effect of rifampicin pre-treatment was superior to rifampicin post-treatment.

Bougainvillea spectabilis flowers extract protects against the rotenone-induced toxicity

Objective:To investigate the effect of two extracts of Bougainvillea spectabilis(B. spectabilis) flowers with yellow and pink/purple on brain oxidative stress and neuronal damage caused in rats by systemic rotenone injection. Methods:Rotenone 1.5 mg/kg was given three times per week alone or in combination with B. spectabilis flowers extracts(25 mg or 50 mg) via the subcutaneous route for 2 weeks. Brain concentrations of the lipid peroxidation marker malondialdehyde(MDA),reduced glutathione,nitric oxide(nitrite),the pro-inflammatory cytokine interleukin-1beta(Il-1β) as well as butyrylcholinesterase,and paraoxonase-1(PON-1) activities,were determined. Histopathology and caspase-3 immunohistochemistry were also performed. Results:Rotenone resulted in significant increases of brain MDA(the product of lipid peroxidation),and nitric oxide content along with decreased brain reduced glutathione. There were also marked and significant inhibition of brain PON-1 and BCh E activities and increased Il-1β in brain of rotenone-treated rats. B. spectabilis flowers extract itself resulted in brain oxidative stress increasing both lipid peroxidation and nitrite content whilst inhibiting PON-1 activity. The yellow flowers extract inhibited BCh E activity and increased brain Il-1β. When given to rotenone-treated rats,B. spectabilis extracts,however,decreased lipid peroxidation while their low administered doses increased brain GSH. Brain nitrite decreased by the pink extract but showed further increase by the yellow extract. Either extract,however,caused further inhibition of PON-1 activity while the yellow extract resulted in further inhibition of BChE activity. Histopathological studies indicated that both extracts protected against brain,liver and kidney damage caused by the toxicant. Conclusions:These data indicate that B. spectabilis flowers extracts exert protective effect against the toxic effects of rotenone on brain,liver and kidney. B. spectabilis flowers extracts decreased brain lipid peroxidation and prevented neuronal death due to rotenone and might thus prove the value in treatment of Parkinson's disease.

Rifampicin inhibits apoptosis in rotenone-induced differentiated PC12 cells by ameliorating mitochondrial oxidative stress

BACKGROUND： Previous studies have shown that rifampicin exhibits neuroprotective effects, but the precise mechanisms remain unclear. Rifampicin is thought to exert the neuroprotective effect as a hydroxyl free radical scavenger. OBJECTIVE： To investigate the protective effects of rifampicin pretreatment on rotenone-induced mitochondrial oxidative stress in differentiated PC12 cells.DESIGN, TIME AND SETrlNG： A repeated measure, cell-based study was performed at the Department of Neurology, Second Affiliated Hospital, Sun Yat-sen University, China between December 2007 and November 2008. MATERIALS： PC12 cells were a kind gift from the Physiology Laboratory of Zhongshan Medical School, Sun Yat-sen University, China. Rotenone and rifampicin were purchased from Sigma, USA. METHODS： PC12 cells were differentiated by culturing with 100 ng/mL 7S nerve growth factor for 9 days in Dulbecco＇s modified Eagle＇s medium/Nutrient Mix F12 （DMEM/F12） supplemented with 10% fetal bovine serum. The cells were assigned to six groups according to various treatment conditions： control, cultured with normal media; rifampicin group, treated with 300 pmol/L rotenone for 26 hours; rotenone group, treated with 2.5 pmol/L rotenone for 24 hours; rifampicin pretreatment groups, pretreated with 100, 200, and 300 pmol/L rifampicin for 2 hours, respectively, followed by 2.5 μmol/L rotenone for 24 hours.MAIN OUTCOME MEASURES： Mitochondrial membrane potential was measured by fluorescence microscopy and flow cytometry, respectively, using rhodamine123 staining. Intracellular reactive oxygen species formation was analyzed by flow cytometry using 2＇, 7＇-dichlorofluorescin-diacetate staining, and intracellular reduced glutathione was measured with a microplate reader. Cell viability was determined by 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide assay. Cell apoptosis was detected by Hoechst 33342 staining and flow cytometry. RESULTS： Increased apoptosis in rotenone-induced, differentiated, PC12 cells was accompanied by the loss of mitochondrial transmembrane potential, the formation of reactive oxygen species, and reduced glutathione depletion （P 〈 0.01）. Rotenone-induced mitochondrial dysfunction was blocked in a dose-dependent manner by rifampicin （P 〈 0.05 or P 〈 0.01）, CONCLUSION： Pretreatment of differentiated PC12 cells with rifampicin blocked rotenone-induced apoptosis by ameliorating mitochondrial dysfunction and oxidative stress.

Differences in brain pathological changes between rotenone and 6-hydroxydopamine Parkinson＇s disease models

Rotenone and 6-hydroxydopamine are two drugs commonly used to generate Parkinson＇s disease animal models.They not only achieve degenerative changes of dopaminergic neurons in the substantia nigra,but also satisfy the requirements for iron deposition.However,few studies have compared the characteristics of these two models by magnetic resonance imaging.In this study,rat models of Parkinson＇s disease were generated by injection of 3 μg rotenone or 10 μg 6-hydroxydopamine into the right substantia nigra.At 1,2,4,and 6 weeks after injection,coronal whole-brain T2-weighted imaging,transverse whole-brain T2-weighted imaging,and coronal diffusion tensor weighted imaging were conducted to measure fractional anisotropy and T2＊ values at the injury site.The fractional anisotropy value on the right side of the substantia nigra was remarkably lower at 6 weeks than at other time points in the rotenone group.In the 6-hydroxydopamine group,the fractional anisotropy value was decreased,but T2＊ values were increased on the right side of the substantia nigra at 1 week.Our findings confirm that the 6-hydroxydopamine-induced model is suitable for studying dopaminergic neurons over short periods,while the rotenone-induced model may be appropriate for studying the pathological and physiological processes of Parkinson＇s disease over long periods.

Detecting dopaminergic neuronal degeneration using diffusion tensor imaging in a rotenone-induced rat model of Parkinson's disease: fractional anisotropy and mean diffusivity values

Dopamine content in the basal ganglia is strongly associated with the degree of dopaminergic neuron loss in the substantia nigra pars com- pacta. Symptoms of Parkinson＇s disease might not arise until more than 50% of the substantia nigra pars compacta is lost and the dopamine content in the basal ganglia is reduced by more than 80%. Greater diagnostic sensitivity and specificity would allow earlier detection of Parkinson＇s disease. Diffusion tensor imaging is a recently developed magnetic resonance imaging technique that measures mean diffusiv- ity and fractional anisotropy, and responds to changes in brain microstructure. When the microscopic barrier （including cell membranes, microtubules and other structures that interfere with the free diffusion of water） is destroyed and extracellular fluid volume accumulates, the mean diffusivity value increases; when the integrity of the microstructure （such as myelin） is destroyed, fractional anisotropy value decreases. However, there is no consensus as to whether these changes can reflect the early pathological alterations in Parkinson＇s disease. Here, we established a rat model of Parkinson＇s disease by injecting rotenone （or sunflower oil in controls） into the right suhstantia nigra. Diffusion tensor imaging results revealed that in the stages of disease, at 1, 2, 4, and 6 weeks after rotenone injection, fiactional anisotropy value decreased, but mean diffusivity values increased in the right substantia nigra in the experimental group. Fractional anisotropy values were lower at 4 weeks than at 6 weeks in the right substantia nigra of rats from the experimental group. Mean diffusivity values were mark- edly greater at 1 week than at 6 weeks in the right corpus striatum of rats from the experimental group. These findings suggest that mean diffusivity and fractional anisotropy values in the brain of rat models of Parkinson＇s disease 4 weeks after model establishment can reflect early degeneration of dopaminergic neurons. ＇The change in fractional anisotropy values after destruction of myelin integrity is likely to be of greater early diagnostic significance than the change in mean diffusivity values.

Mechanisms of rotenone-induced neurotoxicity in PC 12 cells

BACKGROUND： Rotenone-induced neurotoxicity in PC 12 cells has been widely used to study the pathogenesis of Parkinson＇s disease. However, the precise mechanisms underlying rotenone-induced dopaminergic neuronal degeneration in Parkinson＇s disease remains unclear. OBJECTIVE： To establish rotenone-induced neurotoxicity in PC 12 cells, and to investigate the possible action pathways to rotenone-induced neural cell injury at the protein level. DESIGN, TIME AND SETTING： A controlled proteomics study was performed at the Department of Neurology, First Hospital, Jilin University between March 2006 and March 2007. MATERIALS： PC 12 cells were obtained from Shanghai Cell Bank of Chinese Academy of Sciences, China. Rotenone was provided by Sigma, USA. METHODS： PC 12 cells in logarithmic growth phase were treated under experimental and control conditions, respectively. A total of 0.5 μmol/L rotenone, or the same amount of Dulbecco＇s modified eagle＇s medium （DMEM）, was added in the experimental and control conditions, respectively. MAIN OUTCOME MEASURES： Following 72 hours of rotenone treatment, cellular survival rate was determined by methyl thiazolyl tetrazolium assay, and apoptotic changes were detected by Hoechst 33342 staining. Total cellular protein was extracted to acquire differential protein expression data utilizing two-dimensional differential in-gel electrophoresis. To identify differential protein spots, matrix-assisted laser desorption/ionization-time of flight mass spectrometry （MALDI-TOF-MS） was used. RESULTS： In the MTT assay, the experimental condition induced significantly less cell survival compared to the control condition （P 〈 0.01）. Hoechst 33342 staining revealed a larger number of apoptotic cells under the experimental condition compared to the control condition （P 〈 0.01）, as determined by the presence of nuclear condensation, pyknosis, and nuclear fragmentation. Two-dimensional electrophoresis results showed that the differential expression of protein spots 1069 and 1538 was increased by 144% and 124%, respectively, while that of protein spot 1094 was decreased by 123% in the experimental condition compared to the control condition （P 〈 0.01）. By MALDI-TOF-MS analysis and database retrieval, γ-enolase, triosephosphate isomerase 1, and eukaryotic translation initiation factor 4A were confirmed to be involved in rotenone-induced neural cell injury. CONCLUSION： γ-enolase, triosephosphate isomerase 1, and eukaryotic translation initiation factor 4A might participate in rotenone-induced neurotoxicity in PC 12 cells.

Neuroprotection by misoprostol against rotenone-induced neurotoxicity in rat brain

Objective: To investigate the effect of the prostaglandin E1 analogue misoprostol on oxidative stress and neurodegeration caused by subcutaneous rotenone administration in rats. Methods:Rotenone was administered in a dose of 1.5 mg/kg every other day for 2 weeks. Starting from the 1 st day of rotenone injection, rats were subcutaneously treated with misoprostol at doses of10, 100 or 1 000 μg/kg. Rats were evaluated for brain lipid peroxidation(malondialdehyde:MDA), reduced glutathione(GSH), nitric oxide(NO) levels, and paraoxonase-1(PON-1) activity.The concentrations of the anti-apoptotic protein B cell/lymphoma-2(Bcl-2) were determined in the striatum. Histopathologic examination and the expression of inducible nitric oxide synthase(iNOS) in the cerebral cortex and striatum were also performed. Results: Compared with the vehicle-treated group, rotenone caused a significant increase in brain lipid proxidation(MDA)by 61%(P<0.05) accompanied by an increase in NO by 73.1%(P<0.05) and a decrease in GSH concentration by 29.4%(P<0.05). In addition, brain PON-1 activity significantly decreased by63.0%(P<0.05) and striatal Bcl-2 significantly decreased by 27.9%(P<0.05) with respect to the corresponding control value. Brain sections from rotenone treated rats showed extensive dark pyknotic and apoptotic nuclei in neurons, shrunken cytoplasm and perineuronal vacuolation.Rotenone also caused pronounced expression of iNOS in the cerebral cortex and striatum.Treatment with misoprostol at doses of 100 and 1 000 μg/kg resulted in decreased brain MDA(by 16.5%-23.0%)(P<0.05) and NO levels(by 37.1%-40.7%)(P<0.05) and increased GSH concentrations(by 18.8%-30.1%)(P<0.05). PON-1 activity was significantly increased by80.0%-114.8%(P<0.05) by misoprostol at 100 and 1 000 μg/kg, respectively. In addition,misoprostol treatment restored striatal Bcl-2 concentrations to its normal value. Misoprostol treatment resulted in markedly reduced brain injury and decreased iNOS expression in the cerebral cortex and striatum of rotenone intoxicated rats. Conclusions: These data suggest that misoprostol prevents the rotenone-induced neurodegeneration in rat brain by reducing brain oxidative stress.

Neurotoxic effect of rotenone on dopaminergic neuron PC12 in vitro

Objective: To investigate the mechanism of oxidative stress in rotenone neurotoxicity to dopaminergic neuron PC12. Methods: High differentiated PC12 cells as dopaminergic neurons were treated by different concentrations of rotenone. The morphology was observed with inverted phase contrast microscope and transmission electron microscope. Cell viability and proliferation inhibition were assessed by MTT. SOD and MDA were detected with biochemical assay. And the specific fluorescent probe (DCF-DA) was used to examine ROS in PC12 cells. Results: After treated with rotenone for 24 h, most of the PC12 cells became smaller and rounder. The process of axon was reduced, shortened or broken in a time and concentration dependent manner. The mitochondrial structure and metabolism were changed. Endoplasmic reticulum expanded and the free ribosome increased. Compared with the control group, cell proliferation inhibition increased and cell viability decreased. SOD increased and MDA decreased. The intensity of fluorescence was more obvious in PC12 cells treated by rotenone compared with control group. Conclusion : Rotenone is neurotoxic to cultured dopaminergic neuron PC12. Rotenone might exert this effect through the metabolism of oxidative stress on the pathogenesis of the neuron.

Studies on Parkinson’s-Disease-Linked Genes, Brain Urea Levels and Histopathology in Rotenone Induced Parkinson’s Disease Rat Model

Parkinson’s disease (PD) is a debilitating neurological disorder that affects the aged population globally. This study aimed to explore how oral- and intraperitoneal-rotenone-induced PD alters brain urea levels, histopathology, and key Parkinsonism-related genes in the striatum. Hematoxylin and eosin staining was performed for histopathology assessment and real-time polymerase chain reaction was performed for gene expression. Rotenone 3 mg/kg body weight (Rot-3-ip) for 21 days and rotenone 50 mg/kg body weight (Rot-50-po) for 28 days significantly (p < 0.05) altered alpha-synuclein and tyrosine hydroxylase protein expression and Snca, Becn1 and Prkaa1 gene expression in the striatum. Lewy bodies were visible in both Rot-3-ip and Rot-50-po rat brains. There were contrasting features in brain and liver histopathology between the oral and intraperitoneal rotenone treatment groups. However, there was no significant (p < 0.05) difference in the brain urea levels between intraperitoneal and oral rotenone treatment groups. The propagation of PD through oral and intraperitoneal rotenone can have different impacts on the pathological sequence of events based on the molecular approach.

Geniposide prevents rotenone-induced apoptosis in primary cultured neurons

Geniposide, a monomer extracted from gardenia and widely used in Chinese medicine, is a novel agonist at the glucagon-like peptide-1 receptor. This receptor is involved in neuroprotection. In the present study, we sought to identify an anti-apoptotic mechanism for the treatment of neurodegenerative diseases. Primary cultured neurons were treated with different concentrations of rotenone for 48 hours. Morphological observation, cell counting kit-8 assay, lactate dehydrogenase detection and western blot assay demonstrated that 0.5 n M rotenone increased lactate dehydrogenase release, decreased the expression of procaspase-3 and Bcl-2, and increased cleaved caspase-3 expression in normal neurons. All these effects were prevented by geniposide. Our results indicate that geniposide diminished rotenone-induced injury in primary neurons by suppressing apoptosis. This may be one of the molecular mechanisms underlying the efficacy of geniposide in the treatment of neurodegenerative diseases.

The Pael-R gene does not mediate the changes in rotenone-induced Parkinson's disease model cells

In this study, we established cell models for Parkinson＇s disease using rotenone. An RNA interfer- ence vector targeting Parkin-associated endothelin receptor-like receptor （Pad-R） was transfected into the model cells. The results of reverse-transcription polymerase chain reaction and western blot analysis showed that Pael-R expression was decreased after RNA interference compared with the control group （no treatment） and the model group （rotenone treatment）, while the rate of apoptosis and survival of dopaminergic cells did not differ significantly between groups, as detected by flow cytometry and an MTT assay. These experimental findings indicate that the Pael-R gene has no role in the changes in rotenone-induced Parkinson＇s disease model cells.