Protective Effects Of β-asarone On Neurons Damage Induced By Glutamate

Attack rate of nervous system disease upgrades year by year, it threatens the healthy of people severity acutely, to result in heavy burden of society and family, and it becomes the emergent problems to work out a solution for mankind. During the diseases of ischemia brain injured, epilepsy, craniocerebral injury, Parkinson disease and amyotrophic lateral sclerosis, it is the significant cause that neural pathway activation abnormally as well as to bring about excitability toxicity induced by glutamate. Considerable experiments in animal and clinic indicate that it is possible to alleviate neuron injure mediated by glutamate trigger by many cause if glutamate in presynaptic element liberation decrease or glutamic acid receptor in postsynaptic element inhibition. There are a series of glutamic acid receptor antagonist that are confined during clinical application, owing to the existing adverse reactions, even though which show already potential clinical therapeutic effects specifically for many nervous system diseases.Modern research of traditional Chinese medicine (TCM) indicate that a good deal of Chinese crude drug have protection on central nervous system. Our prior study in vivo shows thatβ-asarone could inhibit neuron apoptosis induced by cerebral ischemic-reperfusion injury. For more study and research the protection mechanism ofβ-asarone, to approach amino acids excitability toxicity and corresponding therapeutic medicine, we plan to culture PC12 cells and rat cortical neurons in vitro, then to observe the effect ofβ-asarone on PC12 cells and rat cortical neurons induced by excitatory amino acids——glutamate. We try to interpret excitatory impaired mechanism of glutamate in vivo by this study, to provide elementary study for developing new medicine which could anti-excitatory amino acids toxicity.Objective:①To observe the effects ofβ-asarone on the morphology and cell viability in PC12 cells and rat cortical neurons, then get the initial message aboutβ-asarone interfere in neurons;②To study the protective effects and mechanism of action aboutβ-asarone on PC12 cells and rat cortical neurons damage induced by glutamate.Methods:①Extract volatile oil of Rhizoma Acori Tatarinowii and refiningβ-asarone.②PC12 cells were cultured with basilaris substantia A(there are 3mg Tween-80 and 3mg Glycerine per 100ml medium, which is as same as the composition of 144.057μmol/Lβ-asarone), basilaris substantia B(there are 48mg Tween-80 and 48mg Glycerine per 100ml medium, which is as same as the composition of 2304.912μmol/Lβ-asarone) or 36.014, 72.028, 144.057, 288.114, 576.228, 1152.456, 2304.912μmol/Lβ-asarone for 24h, then to observe morphological changes and cell viability by phase contrast microscope and MTT assay. Established PC12 cells damage model by glutamate.③Established PC12 cells damage model by glutamate.④PC12 cells were cultured with 36.014, 72.028, 144.057μmol/Lβ-asarone for 4h, then cultured with 10mmol/L glutamate for 16h after washing twice, Morphological changes were observed under phase contrast microscope, mitochondrial ultrastructure were observed under transmission electron microscope, cell viability was examined by MTT assay, lactate dehydrogenase(LDH)in culture solution was detected by spectrophotometer, intracellular calcium concentration, apoptosis ratio and mitochondria membrane potential(MMP) were detected by flow cytometry, for assessing the effect onβ-asarone in PC12 cells induced by glutamate.⑤Rat cortical neurocytes were cultured in vitro and stained immunocytochemically with NSE or GFAP antibody respectively on 10th day.⑥Rat cortical neurons were cultured with36.014, 72.028, 144.057, 288.114, 576.228, 1152.45, 2304.912μmol/Lβ-asarone for 24h on 12th to 14th day, then to observe morphological changes and cell viability by phase contrast microscope and MTT assay.⑦Established rat cortical neurons damage model by glutamate.⑧Rat cortical neurons were cultured with36.014, 72.028, 144.057μmol/Lβ-asarone for 4h on 12th to 14th day, then cultured with 40mmol/L glutamate for 16h after washing, morphological changes were observed under phase contrast microscope, mitochondrial ultrastructure were observed under transmission electron microscope, cell viability was examined by MTT assay, LDH in culture solution was detected by spectrophotometer, intracetlular calcium concentration, apoptosis ratio and MMP were detected, by flow cytometry, for assessing the effect onβ-asarone in rat cortical neurons induced by glutamate.Result:①PC12 cells present cellula nervosa's morphous by contrast phase microscope (CPM), neurosomes show triangle, rhombus, there are two to four synapses reciprocal chiasma for reticulate, powerful refraction, round, unitary cellular nucleus were observed by transmission electron microscope(TEM), well-distributed chromoplasm, integrity perinuclear membrane, abundant mitochondria, endocytoplasmic reticulum(ER) and cytolysosome in cytoplasm, integrity cellular membrane, plentiful microvilli, and there were some divided and proliferative PC12 cells which present nucleolemma, intensive segmented chromoplasm.②There are no effects on morphous and cell survival in PC12 cells culutured with basilaris substantia A or basilaris substantia B for 24h, Treatment of PC12 cells with concentrations of 36.014, 72.028, 144.057, 288.114, 576.228, 1152.456, 2304.912μmol/Lβ-asarone for 6h, there were no conspicuous change in PC12 cells cultured with 36.014, 72.028, 144.057, 288.114, 576.228μmol/Lβ-asarone, synapse decreasing, shrinkage, lower refraction, slight cells shedding were observed on PC12 cells cultured with 1152.456, 2304.912μmol/Lβ-asarone. PC12 cells cultured withβ-asarone for 24h, there were no conspicuous change in PC12 cells cultured with 36.014, 72.028, 144.057, 288.114μmol/Lβ-asarone, more impaired cells were observed on PC12 cells cultured with 576.228, 1152.456, 2304.912μmol/Lβ-asarone.③Morphological changes, synapse decreasing, shrinkage, lower refraction, slight cells shedding were observed in PC12 cells exposured to 10mmol/L glutamate for 16h, swollen mitochondrium, degranulation, cristate collapse, expanded ER, cavitation, swollen cytolysosome were observed by TEM. there were no conspicuous change in PC12 cells cultured with 10μmol/L imodipine, 36.014, 72.028,144.057μmol/Lβ-asarone respectively for 4h before exposured to 10mmol/L glutamate for 16h, there are a small quantity PC12 cells cultured with Nimodipine show mitochondrium decreased slightly, cytolysosome minification, spacing appearance, microvilli decreased by TEM, and there were no obviously change of mitochondria ultrastructure in PC12 cells cultured with 144.057μmol/Lβ-asarone.④LDH leakage((607.24±14.31)U/L), apoptosis ratio(10.82%) and intracellular calcium concentration (189.29±19.72) increasing, cell survival(0.88±0.05) and MMP(compare to control, (66.88±4.70)%) decreasing in PC12 cells exposured to 10mmol/L glutamate. PC12 cells cultured with 10μmol/L Nimodipine, 36.014, 2.028,44.057μmol/Lβ-asarone respectively for 4h before exposured to 10mmol/L glutamate, their cell survival, LDH leakage, apoptosis ratio, intracellular calcium concentration and MMP respectively are (1.00±0.02, 1.00±0.04, 0.97±0.04, 0.94±0.01), ((272.46±22.02, 375.44±15.07, 519.08±11.07, 550.24±13.62)U/L), (4.90±1.14, 4.95±1.84, 6.83±1.79, 8.68±1.37)%, (144.73±14.02, 145.73±21.25, 158.99±28.12, 176.93±18.31), (88.28±1.55, 81.36±6.19, 77.14±6.58, 70.36±6.90).⑤Perikaryons under CPM present metuliform or polygon, boles and branches of synapses extended and thickening obviously, conspicuous halation, refraction strengthen, to form crowded network. Immunocytochemical test shows that most of the cultured rat cortical neurocytes were positively stained with neuron specific enolase (NSE) antibody, and positively with glial fibrillary acidic protein (GFAP) in a less degree. Cubic capacity of neurons, is larger, cellular nucleus and cytoplasm is bigger comparatively, light gray, well-distributed chromoplasm, integrated double-deck nuclear membrane, abundant mitochondria, cytolysosome and ER were observed by TEM.⑥Rat cortical neurons were cultured with36.014, 72.028, 144.057, 288.114, 576.228, 1152.456, 2304.912μmol/Lβ-asarone for 24h on 12th to 14th day, there were no conspicuous change in neurons cultured with 36.014, 72.028, 144.057, 288.114, 576.228μmol/Lβ-asarone, compare to control, perikaryons of neurons cultured with l152.456μmol/Lβ-asaron are larger, impaired cells were observed on neurons cultured with 2304.912μmol/Lβ-asarone.⑦Neurons on 12th to 14th day exposured to 40mmol/L glutamate for 16h, lower outstanding refraction, parts cell rounding, shrinkage, defluxion, flagment under CPM; Round neclei, smooth completed nuclear membrane, aggregate chromoplasm, swollen cytomicrosome, expanded ER were observed by TEM. There were no conspicuous change under CPM in neurons on 12th to 14th day cultured with 10μmol/L imodipine, 36.014, 72.028, 144.057μmol/Lβ-asarone respectively for 4h before exposured to 40mmol/L glutamate for 16h, no much ultramicrostructure change expect small amounts aggregate chromoplasm, swollen cytomicrosom and expanded ER slightly were observed by TEM in neurons cultured with 144.057μmol/Lβ-asarone for 4h before exposured to 40mmol/L glutamate for 16h.⑧LDH leakage((619.40±36.71)U/L), apoptosis ratio((21.05±4.81)%) and intracellular calcium concentration((107.49±21.10)) increasing, cell survival(0.45±0.01) and MMP((91.93±8.54)%, compare to the control, (P＞0.05)) decreasing were observed in cultured rat cortical neurons on 12th to 14th day exposured to 40mmol/L glutamate for 16, rat cortical neurons cultured with 10μmol/L Nimodipine and 36.014, 2.028, 44.057μmol/Lβ-asarone respectively for 4h before exposured to 40mmol/L glutamate, their cell survival, LDH leakage, apoptosis ratio, intracellular calcium concentration and MMP respectively are (0.66±0.01, 0.63±0.01, 0.58±0.01, 0.56±0.01), (440.80±27.28, 475.00±33.51, 561.64±39.36, 593.56±41.50)U/L, (6.80±2.48, 5.97±0.88, 9.01±2.46, 9.83±1.92)%, (92.56±21.13, 94.64±20.97, 84.89±18.82, 107.57±24.12), (99.27±1.89, 98.57±2.48, 95.48±7.22, 95.10±5.16)%。Conclusion:①Differentiated PC12 cells were cultured in vitro, the vaccination containing 5% fetal bovine serum, 5% horse serum and 95% high-glucose DMEM medium. Small triangles were observed and adherence completely on 2th hour, then, they show nerve-like cells on 12th hour, cells bodies present roughly triangular, diamond shape, spindle, stretched out 2 to 4 synapsis, strong refraction. synapsis protrude longer, criss-cross on 24th hour, PC12 cells show reticulodromous on 48th hour, and the morphology was consistent with nerve cells.②There are no effects on morphous and cell survival in PC12 cells culutured with basilaris substantia A or basilaris substantia B for 6 hours or 24 hours, 576.228, 1152.456, 2304.912μmol/Lβ-asarone can inhibit the proliferation of PC12 cells.β-asarone inhibit the proliferation of PC12 cells which is a link role in the cell cycle and the mechanism through which to achieve, we still need further study.③The toxic effect of glutamate on PC12 cells shows in a dose-dependent relationship, toxicity enhanced with the concentration of glutamate increasing. PC12 cells were cultured with 10mmol/L glutamate for 16h, apoptosis was observed.④PC12 cells were cultured with 36.014, 72.028, 144.057μmol/Lβ-asarone respectively for 4 hours before induced by 10mmol/L glutamate,β-asarone can reduce the damage of PC12 induced by glutamate by inhibiting LDH leakage, reducing abnormal elevation of intracellular calcium ion concentration and stablizing MMP state, thus playing the role of anti-neuronal apoptosis.⑤High purity rat cortical neurons can be obtained by this cultured method in this study, which facilitates revealing neurons, ultrastructural neural networks, neuronal, biochemical and pathological changes in the complex neural pharmacology research.⑥Rat cerebral cortical neurons cultured withβ-asarone within normal limit are promoted, nerve cell bodies larger, intensive synapsis.⑦The toxic effect of glutamate on cortical neurons shows in a dose-dependent relationship, toxicity enhanced with the concentration of glutamate increasing. Rat cortical neurons were cultured with 40mmol/L glutamate for 16h, neuronal apoptosis was observed.⑧Rat cortical neurons were cultured with 36.014, 72.028, 144.057μmol/Lβ-asarone respectively for 4 hours before induced by 40mmol/L glutamate,β-asarone can reduce the damage of neurons induced by glutamate, inhibit LDH leakage, reduce abnormal elevation of intracellular calcium ion concentration, but no significant effect on MMP, The effect ofβ-asarone on anti-neuronal apoptosis maybe attribute to the inhibition of glutamate receptors activity and anticalcium.