β-细辛醚对谷氨酸所致神经元损伤的保护作用
摘要
神经系统疾病发病率逐年上升，严重威胁着人们的健康，造成沉重的社会及经济负担，已成为人类迫切需要解决的首要问题。在缺血性中风、癫痫、颅脑损伤、帕金森病、肌萎缩侧索硬化等疾病过程中，谷氨酸能神经通路异常激活并由此而产生的兴奋性毒性，是造成神经元损伤的重要原因。已有大量动物实验研究和逐渐增多的临床实验表明，能够减少突触前谷氨酸释放，或抑制突触后谷氨酸受体的物质，有可能减轻多种原因触发的谷氨酸介导的神经元损伤。尽管目前已经明确和开发了一系列的谷氨酸受体拮抗剂，对多种神经系统疾病有潜在的临床应用价值，但由于其存在的不良反应而限制了其在临床方面的应用。
中医药的现代研究表明很多中药对中枢神经系统有保护作用，本实验室前期在体实验研究结果表明β-细辛醚可显著抑制由脑缺血——再灌注诱导的神经细胞凋亡。为了更加深入地研究和明确β-细辛醚的作用机制，探讨神经系统疾病中起主要作用的氨基酸兴奋性毒性机制及其治疗药物，本实验拟在体外培养PC12细胞和原代培养乳鼠脑皮层神经元的基础上，探讨β-细辛醚对兴奋性氨基酸——谷氨酸损伤神经元的干预作用，试图通过本研究，能够阐明体外谷氨酸兴奋性损伤的作用机制及为开发抗兴奋性氨基酸毒性新药提供研究基础。
研究目的：
1．观察β-细辛醚对生理状态下PC12细胞和大鼠脑皮层神经元形态和细胞活力的影响，初步了解β-细辛醚对神经细胞的干预作用；
2．观察β-细辛醚对谷氨酸诱导的PC12细胞及乳鼠脑皮层神经元的保护作用，并探讨其作用机理。
研究方法：
1．提取并精制β-细辛醚；
2．将基质A(每100ml培养基中含3mg吐温-80和3mg甘油)、基质B(每100ml培养基中含48mg吐温-80和48mg甘油)及36.014、72.028、144.057、288.114、576.228、1152.456、2304.912μmol／Lβ-细辛醚与PCI2细胞共培养24h，观察对其形态学和细胞活力的影响；
3．建立PC12细胞谷氨酸损伤模型；
4．体外培养PC12细胞，β-细辛醚(36.014、72.028、144.057μmol／L)预给药4h，荡洗后加入终浓度为10mmol／L的谷氨酸继续培养16h，MTT法检测细胞活力，倒置相差显微镜观察细胞形态学变化，透射电镜观察细胞超微结构改变，分光光度计检测培养上清中乳酸脱氢酶(LDH)的含量，流式细胞术检测细胞凋亡率、细胞内游离钙离子浓度、线粒体膜电位(MMP)的变化，探讨β-细辛醚对谷氨酸诱导损伤的PC12细胞的干预作用；
5．体外培养大鼠脑皮层神经元，培养至第10d，光学显微镜和免疫细胞化学染色鉴定神经元；
6．建立大鼠脑皮层神经元谷氨酸损伤模型；
7．将36.014、72.028、144.057、288.114、576.228、1152.456、2304.912μmol／Lβ-细辛醚与培养至第12-14d的脑皮层神经元共培养24h，观察对其形态学和细胞活力的影响；
8．脑皮层神经元培养至第12～14d，β-细辛醚(36.014、72.028、144.057μmol／L)预给药4h，荡洗后加入终浓度为40mmol／L的谷氨酸继续培养16h，MTT法检测细胞活力，应用倒置相差显微镜观察细胞形态学变化，透射电镜观察细胞超微结构改变，分光光度计检测培养上清中LDH的含量，流式细胞仪检测神经元凋亡率、神经元胞内游离钙离子浓度、MMP的变化，探讨β-细辛醚对谷氨酸诱导损伤的脑皮层神经元的干预作用。
研究结果：
1．相差显微镜下PC12细胞呈现神经细胞样形态，胞体呈三角形、菱形、梭形，2～4个突触，相互交叉成网状，折光性较强：透射电镜下细胞核呈圆形，单个存在，染色质分布均匀，核膜完整，细胞质富含线粒体、内质网、溶酶体，细胞膜完整，微绒毛丰富；也可见核膜消失，染色质密集，成分叶状，为处于分裂、增殖的PC12细胞；
2．PC12细胞与基质A组、基质B组及36.014、72.028、144.057、288.114、576.228、1152.456、2304.912μmol／Lβ-细辛醚共培养6h，基质A组、基质B组及36.014～576.228μmol／Lβ-细辛醚组细胞形态未有明显改变，1152.456、2304.912μmol／Lβ-细辛醚组细胞的突起减少，细胞皱缩，折光性下降，部分细胞脱落；共培养24h，基质A组、基质B组及36.014～288.114μmol／Lβ-细辛醚组PC12细胞形态未有明显改变，576.228μmol／Lβ-细辛醚组PC12细胞已有损伤，表现为细胞突起结构消失减少、细胞圆缩聚集，1152.456、2304.912μmol／Lβ-细辛醚组PC12细胞损伤进一步加重；
3．10mmol／L的谷氨酸与PC12细胞共培养16h，相差显微镜下见细胞不同程度的变圆、皱缩，突触缩短、减少，局部脱落、裂解成碎片；透射电镜未见明显凋亡特征性改变，细胞核完整，染色质减少、溶解消失，出现空隙，细胞质疏松，线粒体肿胀、脱颗粒、嵴断裂，空泡形成，内质网扩张、形成空泡，溶酶体肿胀，细胞膜尚完整，微绒毛消失。10μmol／L尼莫地平及36.014、72.028、144.057μmol／Lβ-细辛醚预给药4h，光镜下尼莫地平组及β-细辛醚组细胞形态无明显改变，透射电镜下可见尼莫地平组少量细胞线粒体略减少，溶酶体缩小，出现空隙，微绒毛减少等改变，也可见与正常培养PC12细胞结构无明显差异的细胞；144.057μmol／Lβ-细辛醚组细胞超微结构无明显改变；
4．10mmol／L的谷氨酸与PC12细胞共培养16h，细胞活力下降(0.88±0.05)，培养液中LDH含量增多((607.24±14.31)U／L)，细胞凋亡率增加(10.82％)，细胞内游离钙离子浓度升高(189.29±19.72)，线粒体膜电位下降((66.88±4.70)％)；10μmol／L尼莫地平组及144.057、72.028、36.014μmol／Lβ-细辛醚组的细胞活力、培养液中LDH含量、细胞凋亡率、细胞内钙离子浓度及线粒体膜电位分别为(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)％；
5．相差显微镜下培养至10d的脑皮层神经元胞体呈锥形或多角形，突起主干和分支明显延长并增粗，有明显的光晕，折光性增强，富有立体感，形成稠密的网络结构；细胞免疫化学染色结果显示，大部分细胞可见神经元烯醇酶(neuron specific enolase，NSE)免疫阳性颗粒位于核周质及突起中(呈棕黄色)，细胞核不染色，为一圆形或椭圆形淡染区，胶质纤维酸性蛋白(glial fibrillary acidic protein，GFAP)染色呈弱阳性；透射电镜可见神经元体积较大，细胞核、细胞质均较大，呈浅灰色着色，染色质分布均匀，核膜双层结构完整，线粒体、溶酶体、内质网等细胞器较丰富；.6．培养至12～14d的脑皮层神经元与36.014、72.028、144.057、288.114、576.228、1152.456、2304.912μmol／Lβ-细辛醚共培养24h，与正常培养的神经元比较，36.014～576.228μmol／Lβ-细辛醚组神经元形态无明显改变；而1152.456μmol／Lβ-细辛醚组神经元胞体相对较大；2304.912μmol／Lβ-细辛醚则对神经元有损伤作用，神经元变圆、皱缩，折光性下降，部分突起收缩；
7．培养至12～14d的脑皮层神经元与40mmol／L谷氨酸共培养16h后，折光性明显下降，部分细胞变圆、皱缩、脱落，裂解成碎片，透射电镜可见神经元胞核呈圆形，核膜光滑、完整，染色质边聚，线粒体肿胀，部分内质网扩张；10μmol／L尼莫地平及36.014、72.028、144.057μmol／Lβ-细辛醚预给药4h，能不同程度地减轻谷氨酸对脑皮层神经元的损伤，光镜下细胞形态无明显改变，透射电镜下可见144.057μmol／Lβ-细辛醚组其神经元除少量染色质边聚、内质网轻微扩张、线粒体轻度肿胀外，余与正常培养的脑皮层神经元无明显差异；.8．培养至12～14d的脑皮层神经元与40mmol／L谷氨酸共培养16h后，其凋亡细胞增多(21.05±4.81)％，培养液中LDH含量增高(619.40±36.71)U／L，细胞内游离钙离子浓度升高(107.49±21.10)，线粒体膜电位轻微下降(91.93±8.54)％，但与正常对照组比较，未有统计学差异(P>0．05)；1μmol／L尼莫地平组及144.057、72.028、36.014μmol／Lβ-细辛醚组的细胞活力、培养液中LDH含量、细胞凋亡率、细胞内钙离子浓度、线粒体膜电位分别为(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)％。
研究结论：
1．本实验体外培养高分化PC12细胞，接种在含5％胎牛血清、5％马血清的高糖DMEM培养基培养，2h后完全贴壁，细胞呈小三角形，12h呈现神经细胞样形态，胞体呈三角形、菱形、梭形，伸出2～4个突起，折光性较强，24h后突起较长，互相交叉，48h后细胞生长呈网状，符合神经细胞的形态结构，可用于实验；
2．基质A、基质B对正常培养的PC12细胞的形态学及细胞活力无明显影响，576.228、1152.456、2304.912μmol／L的β-细辛醚能抑制体外培养的PC12细胞增殖。关于β-细辛醚对体外培养的PC12细胞增殖的抑制作用是作用于增殖周期中的哪一个环节，并通过何种机制来实现，还需要进一步研究；
3．谷氨酸对高分化PC12细胞的毒性作用有明显的剂量依赖关系，随着谷氨酸浓度的增加，其毒性作用增强；10mmol／L谷氨酸与PC12细胞共培养16h，可造成PC12细胞凋亡模型；
4．36.014、72.028、144.057μmol／Lβ-细辛醚预给药4h，均能不同程度地减轻10mmol／L谷氨酸对PC12的损伤作用，抑制LDH漏出，拮抗细胞内钙离子浓度的异常升高，稳定MMP状态，从而发挥其抗神经元凋亡的作用；
5．本实验建立的新生大鼠脑皮层神经元培养方法，可得到纯度较高的神经元，为揭示神经元、神经网络超微结构及神经元生理、生化和病理等方面的复杂变化和研究神经药理学提供了便利；
6．一定浓度的β-细辛醚对正常培养的大鼠脑皮层神经元有促生长作用，其神经元胞体较大，突起密集；
7．谷氨酸对脑皮层神经元的毒性作用有明显的剂量依赖关系，随着谷氨酸浓度的增加，其毒性作用增强；40mmol／L谷氨酸与脑皮层神经元共培养16h，可造成神经元凋亡模型；
8．36.014、72.028、144.057μmol／Lβ-细辛醚预给药4h，均能不同程度地减轻经40mmol／L谷氨酸诱导损伤的脑皮层神经元其凋亡和坏死，抑制LDH漏出，减少由谷氨酸引起的细胞内钙离子异常增多，但对MMP无明显影响。表明β-细辛醚可能通过抑制谷氨酸受体活性、钙拮抗作用而减轻谷氨酸诱导的神经元的损伤。
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.
中医药的现代研究表明很多中药对中枢神经系统有保护作用，本实验室前期在体实验研究结果表明β-细辛醚可显著抑制由脑缺血——再灌注诱导的神经细胞凋亡。为了更加深入地研究和明确β-细辛醚的作用机制，探讨神经系统疾病中起主要作用的氨基酸兴奋性毒性机制及其治疗药物，本实验拟在体外培养PC12细胞和原代培养乳鼠脑皮层神经元的基础上，探讨β-细辛醚对兴奋性氨基酸——谷氨酸损伤神经元的干预作用，试图通过本研究，能够阐明体外谷氨酸兴奋性损伤的作用机制及为开发抗兴奋性氨基酸毒性新药提供研究基础。
研究目的：
1．观察β-细辛醚对生理状态下PC12细胞和大鼠脑皮层神经元形态和细胞活力的影响，初步了解β-细辛醚对神经细胞的干预作用；
2．观察β-细辛醚对谷氨酸诱导的PC12细胞及乳鼠脑皮层神经元的保护作用，并探讨其作用机理。
研究方法：
1．提取并精制β-细辛醚；
2．将基质A(每100ml培养基中含3mg吐温-80和3mg甘油)、基质B(每100ml培养基中含48mg吐温-80和48mg甘油)及36.014、72.028、144.057、288.114、576.228、1152.456、2304.912μmol／Lβ-细辛醚与PCI2细胞共培养24h，观察对其形态学和细胞活力的影响；
3．建立PC12细胞谷氨酸损伤模型；
4．体外培养PC12细胞，β-细辛醚(36.014、72.028、144.057μmol／L)预给药4h，荡洗后加入终浓度为10mmol／L的谷氨酸继续培养16h，MTT法检测细胞活力，倒置相差显微镜观察细胞形态学变化，透射电镜观察细胞超微结构改变，分光光度计检测培养上清中乳酸脱氢酶(LDH)的含量，流式细胞术检测细胞凋亡率、细胞内游离钙离子浓度、线粒体膜电位(MMP)的变化，探讨β-细辛醚对谷氨酸诱导损伤的PC12细胞的干预作用；
5．体外培养大鼠脑皮层神经元，培养至第10d，光学显微镜和免疫细胞化学染色鉴定神经元；
6．建立大鼠脑皮层神经元谷氨酸损伤模型；
7．将36.014、72.028、144.057、288.114、576.228、1152.456、2304.912μmol／Lβ-细辛醚与培养至第12-14d的脑皮层神经元共培养24h，观察对其形态学和细胞活力的影响；
8．脑皮层神经元培养至第12～14d，β-细辛醚(36.014、72.028、144.057μmol／L)预给药4h，荡洗后加入终浓度为40mmol／L的谷氨酸继续培养16h，MTT法检测细胞活力，应用倒置相差显微镜观察细胞形态学变化，透射电镜观察细胞超微结构改变，分光光度计检测培养上清中LDH的含量，流式细胞仪检测神经元凋亡率、神经元胞内游离钙离子浓度、MMP的变化，探讨β-细辛醚对谷氨酸诱导损伤的脑皮层神经元的干预作用。
研究结果：
1．相差显微镜下PC12细胞呈现神经细胞样形态，胞体呈三角形、菱形、梭形，2～4个突触，相互交叉成网状，折光性较强：透射电镜下细胞核呈圆形，单个存在，染色质分布均匀，核膜完整，细胞质富含线粒体、内质网、溶酶体，细胞膜完整，微绒毛丰富；也可见核膜消失，染色质密集，成分叶状，为处于分裂、增殖的PC12细胞；
2．PC12细胞与基质A组、基质B组及36.014、72.028、144.057、288.114、576.228、1152.456、2304.912μmol／Lβ-细辛醚共培养6h，基质A组、基质B组及36.014～576.228μmol／Lβ-细辛醚组细胞形态未有明显改变，1152.456、2304.912μmol／Lβ-细辛醚组细胞的突起减少，细胞皱缩，折光性下降，部分细胞脱落；共培养24h，基质A组、基质B组及36.014～288.114μmol／Lβ-细辛醚组PC12细胞形态未有明显改变，576.228μmol／Lβ-细辛醚组PC12细胞已有损伤，表现为细胞突起结构消失减少、细胞圆缩聚集，1152.456、2304.912μmol／Lβ-细辛醚组PC12细胞损伤进一步加重；
3．10mmol／L的谷氨酸与PC12细胞共培养16h，相差显微镜下见细胞不同程度的变圆、皱缩，突触缩短、减少，局部脱落、裂解成碎片；透射电镜未见明显凋亡特征性改变，细胞核完整，染色质减少、溶解消失，出现空隙，细胞质疏松，线粒体肿胀、脱颗粒、嵴断裂，空泡形成，内质网扩张、形成空泡，溶酶体肿胀，细胞膜尚完整，微绒毛消失。10μmol／L尼莫地平及36.014、72.028、144.057μmol／Lβ-细辛醚预给药4h，光镜下尼莫地平组及β-细辛醚组细胞形态无明显改变，透射电镜下可见尼莫地平组少量细胞线粒体略减少，溶酶体缩小，出现空隙，微绒毛减少等改变，也可见与正常培养PC12细胞结构无明显差异的细胞；144.057μmol／Lβ-细辛醚组细胞超微结构无明显改变；
4．10mmol／L的谷氨酸与PC12细胞共培养16h，细胞活力下降(0.88±0.05)，培养液中LDH含量增多((607.24±14.31)U／L)，细胞凋亡率增加(10.82％)，细胞内游离钙离子浓度升高(189.29±19.72)，线粒体膜电位下降((66.88±4.70)％)；10μmol／L尼莫地平组及144.057、72.028、36.014μmol／Lβ-细辛醚组的细胞活力、培养液中LDH含量、细胞凋亡率、细胞内钙离子浓度及线粒体膜电位分别为(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)％；
5．相差显微镜下培养至10d的脑皮层神经元胞体呈锥形或多角形，突起主干和分支明显延长并增粗，有明显的光晕，折光性增强，富有立体感，形成稠密的网络结构；细胞免疫化学染色结果显示，大部分细胞可见神经元烯醇酶(neuron specific enolase，NSE)免疫阳性颗粒位于核周质及突起中(呈棕黄色)，细胞核不染色，为一圆形或椭圆形淡染区，胶质纤维酸性蛋白(glial fibrillary acidic protein，GFAP)染色呈弱阳性；透射电镜可见神经元体积较大，细胞核、细胞质均较大，呈浅灰色着色，染色质分布均匀，核膜双层结构完整，线粒体、溶酶体、内质网等细胞器较丰富；.6．培养至12～14d的脑皮层神经元与36.014、72.028、144.057、288.114、576.228、1152.456、2304.912μmol／Lβ-细辛醚共培养24h，与正常培养的神经元比较，36.014～576.228μmol／Lβ-细辛醚组神经元形态无明显改变；而1152.456μmol／Lβ-细辛醚组神经元胞体相对较大；2304.912μmol／Lβ-细辛醚则对神经元有损伤作用，神经元变圆、皱缩，折光性下降，部分突起收缩；
7．培养至12～14d的脑皮层神经元与40mmol／L谷氨酸共培养16h后，折光性明显下降，部分细胞变圆、皱缩、脱落，裂解成碎片，透射电镜可见神经元胞核呈圆形，核膜光滑、完整，染色质边聚，线粒体肿胀，部分内质网扩张；10μmol／L尼莫地平及36.014、72.028、144.057μmol／Lβ-细辛醚预给药4h，能不同程度地减轻谷氨酸对脑皮层神经元的损伤，光镜下细胞形态无明显改变，透射电镜下可见144.057μmol／Lβ-细辛醚组其神经元除少量染色质边聚、内质网轻微扩张、线粒体轻度肿胀外，余与正常培养的脑皮层神经元无明显差异；.8．培养至12～14d的脑皮层神经元与40mmol／L谷氨酸共培养16h后，其凋亡细胞增多(21.05±4.81)％，培养液中LDH含量增高(619.40±36.71)U／L，细胞内游离钙离子浓度升高(107.49±21.10)，线粒体膜电位轻微下降(91.93±8.54)％，但与正常对照组比较，未有统计学差异(P>0．05)；1μmol／L尼莫地平组及144.057、72.028、36.014μmol／Lβ-细辛醚组的细胞活力、培养液中LDH含量、细胞凋亡率、细胞内钙离子浓度、线粒体膜电位分别为(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)％。
研究结论：
1．本实验体外培养高分化PC12细胞，接种在含5％胎牛血清、5％马血清的高糖DMEM培养基培养，2h后完全贴壁，细胞呈小三角形，12h呈现神经细胞样形态，胞体呈三角形、菱形、梭形，伸出2～4个突起，折光性较强，24h后突起较长，互相交叉，48h后细胞生长呈网状，符合神经细胞的形态结构，可用于实验；
2．基质A、基质B对正常培养的PC12细胞的形态学及细胞活力无明显影响，576.228、1152.456、2304.912μmol／L的β-细辛醚能抑制体外培养的PC12细胞增殖。关于β-细辛醚对体外培养的PC12细胞增殖的抑制作用是作用于增殖周期中的哪一个环节，并通过何种机制来实现，还需要进一步研究；
3．谷氨酸对高分化PC12细胞的毒性作用有明显的剂量依赖关系，随着谷氨酸浓度的增加，其毒性作用增强；10mmol／L谷氨酸与PC12细胞共培养16h，可造成PC12细胞凋亡模型；
4．36.014、72.028、144.057μmol／Lβ-细辛醚预给药4h，均能不同程度地减轻10mmol／L谷氨酸对PC12的损伤作用，抑制LDH漏出，拮抗细胞内钙离子浓度的异常升高，稳定MMP状态，从而发挥其抗神经元凋亡的作用；
5．本实验建立的新生大鼠脑皮层神经元培养方法，可得到纯度较高的神经元，为揭示神经元、神经网络超微结构及神经元生理、生化和病理等方面的复杂变化和研究神经药理学提供了便利；
6．一定浓度的β-细辛醚对正常培养的大鼠脑皮层神经元有促生长作用，其神经元胞体较大，突起密集；
7．谷氨酸对脑皮层神经元的毒性作用有明显的剂量依赖关系，随着谷氨酸浓度的增加，其毒性作用增强；40mmol／L谷氨酸与脑皮层神经元共培养16h，可造成神经元凋亡模型；
8．36.014、72.028、144.057μmol／Lβ-细辛醚预给药4h，均能不同程度地减轻经40mmol／L谷氨酸诱导损伤的脑皮层神经元其凋亡和坏死，抑制LDH漏出，减少由谷氨酸引起的细胞内钙离子异常增多，但对MMP无明显影响。表明β-细辛醚可能通过抑制谷氨酸受体活性、钙拮抗作用而减轻谷氨酸诱导的神经元的损伤。
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.
引文
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