颅脑创伤后亚低温脑保护的蛋白质组研究

目的 本研究应用差异蛋白质组学技术,探讨亚低温和常温条件下,创伤性颅脑损伤大鼠海马组织蛋白质的表达变化.方法 采用侧方液压冲击装置,建立大鼠中度脑损伤模型,亚低温组(n=3)于伤后维持体温(33±0.5)℃持续3h,常温组(n=3)始终维持体温(37±0.5)℃.取大鼠海马组织,通过差异荧光双向凝胶电泳、分离蛋白,获得二维的蛋白质分离图谱,然后通过胶内酶切、抽提酶解肽段、基质辅助激光解吸飞行时间质谱分析差异的蛋白质点,鉴定出变化的蛋白质.结果 通过DeCyder5.0(GE Healthcare)软件分析报告发现差异1.5倍以上的蛋白点17个(P＜0.05).通过对这些蛋白考染点进行质谱鉴定,鉴定出14个蛋白质,2个为同一种蛋白,实际差异蛋白数为13个.分别是细胞骨架蛋白、介导能量代谢的酶类、参与核酸合成、氧化应激反应的蛋白质、神经突触功能蛋白、细胞内信号传递蛋白及未知蛋白.结论 脑损伤后亚低温及常温条件下,大鼠海马组织蛋白质存在表达差异,鉴定出的差异蛋白质可能与亚低温保护效应的潜在作用机制有关.     

海人酸颞叶癫痫大鼠海马组织中细胞骨架蛋白的差异表达

目的 研究海人酸颞叶癫痫大鼠海马组织中差异表达的细胞骨架蛋白,为阐明癫痫的发病机制提供线索,为新型抗癫痫药物的研发提供分子靶点.方法 利用双向荧光差异凝胶电泳(2D-DIGE)、DeCyder分析软件、基质辅助激光解吸附电离串联飞行时间质谱(MALDI-TOF/TOF-MS)对海人酸诱导颞叶癫痫大鼠海马组织中的差异蛋白进行分离、分析及鉴定.结果 有5个差异蛋白点被鉴定为3种细胞骨架蛋白,其中微管蛋白α-1B和胶质纤维酸性蛋白表达上调,埃兹蛋白表达下调.结论 细胞骨架蛋白表达变化导致的细胞骨架受损可能与癫痫的发生发展相关,可能成为抗癫痫治疗新靶点.     

脑血管痉挛中差异表达蛋白质的实验研究

目的 建立兔脑血管痉挛(CVS)基底动脉和正常兔基底动脉双向凝胶电泳图谱,鉴定差异表达蛋白,从中发现有意义的CVS相关标志物.方法 采用双向凝胶电泳(2-DE)分离痉挛基底动脉和正常基底动脉总蛋白,银染显色,通过imagemaster5.0软件分析,从中选取差异表达蛋白质点,应用基质辅助激光解析飞行时间串联质谱(MALDI-TOF/TOF)鉴定差异表达的蛋白质.结果 获得重复性和分辨率较好的兔基底动脉双向凝胶电泳图谱;发现49个差异表达点,其中35个点得到鉴定,在CVS中高表达的有24个,其余11个在CVS中呈低表达.结论 建立了痉挛基底动脉和正常基底动脉双向凝胶电泳图谱,并应用质谱技术鉴定了35个差异表达点,这些差异表达蛋白质可能与CVS的发生相关.     

创伤性脑损伤大鼠海马蛋白质差异表达研究

目的 应用蛋白质组学方法分析研究大鼠创伤性脑损伤后海马组织差异表达蛋白质,为筛选脑损伤早期诊断的生物学标志物提供理论依据.方法 采用双向凝胶电泳分离总蛋白,硝酸银染色,PDQuest 7.0图像分析软件分析获得的差异蛋白质点,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)对检测到的差异蛋白质点进行鉴定,获得肽质量指纹图谱,查阅蛋白质数据库得到所需蛋白质相关信息,分析大鼠创伤性脑损伤后4 h、8 h、12 h、24 h和48 h海马组织中差异表达蛋白质,每组样品电泳重复3次,每帧图谱检测到1800余个蛋白质点;选择2个背景清晰、重复性及分辨力良好、在各组表达差异明显并呈现时序性变化的蛋白质点进行质谱分析.结果 经双向凝胶电泳分离共获得18帧图,每组3帧,其中对照组及损伤后12 h组蛋白质图谱显示分离效果良好,蛋白质点清晰,点染色程度较深,数目较多.横纹和纵纹较少,图谱平均匹配率>80%.采用PDQuest 7.0图像分析软件对蛋白质图谱进行定量分析证实,相对分子质量为58.00×103的差异蛋白质为葡萄糖调节蛋白(葡萄糖调节蛋白58),在损伤后4 h、8 h和12 h表达水平上调;相对分子质量为28.40×103的差异蛋白质为蛋白酶体α亚单位3型.在损伤后4 h、8 h、12 h、24 h和48 h表达水平均上调.结论 葡萄糖调节蛋白58和蛋白酶体α亚单位3型可能与创伤性脑损伤的发生、发展密切相关,此为探讨脑损伤的发病机制提供了重要线索.     

耐药性杏仁核点燃癫痫模型海马组织中γ-氨基丁酸受体表达变化

目的 建立多药耐药性癫痫模型,观察海马组织γ-氨基丁酸(GABA)受体表达变化从而探讨其在耐药性杏仁核点燃癫痫形成中的作用.方法 选用Wistar大鼠100只制作慢性杏仁核点燃癫痫模型,模型制作成功(n=52)后用经典抗癫痫药苯妥英钠和苯巴比妥进行筛选,根据大鼠对苯妥英钠和苯巴比妥的反应区别出耐药癫痫大鼠(n=8)及药物敏感大鼠(n=8),然后处死动物留取脑组织标本,用免疫组织化学染色方法观察海马组织内GABAA受体表达变化,用蛋白质印迹法检测GABAA受体含量,观察耐药癫痫大鼠和药物敏感大鼠之间的不同.结果 耐药癫痫性颞叶大鼠海马细胞变性坏死,排列紊乱,结构特征消失;耐药性颞叶癫痫大鼠海马组织内GABAA受体阳性表达细胞的灰度值(141.15±14.72)比药物敏感大鼠增高(92.56 ±5.17;t =3.380,P=0.006);蛋白质印迹方法提示受体条带变淡变窄,蛋白含量明显减少(0.38 ±0.08),与药物敏感大鼠(0.88 ±0.18)比较,差异具有统计学意义(t=5.420,P=0.002);但两组间GABAA受体阳性细胞数百分率比较差异无统计学意义.结论 耐药性颞叶癫痫大鼠海马组织内GABA受体表达明显减少,这可能在耐药性颞叶癫痫的形成过程中发挥部分作用.     

SELDI-TOF-MS和双向凝胶电泳技术联用分析脑损伤后海马组织蛋白质表达谱的变化

目的 分析大鼠闭合性脑损伤后海马中蛋白质表达的变化及特点。 方法 选成年雄性SD大鼠随机分为手术对照组及损伤后不同时间点组, 复制Marmarou 落体打击闭合性颅脑损伤模型,应用蛋白质芯片技术和双向凝胶电泳技术分析大鼠脑损伤后海马中蛋白质表达的改变。并选择2个背景清晰、重复性及分辨率较好、在各组蛋白表达差异明显并呈现时序性变化的点进行质谱分析。结果 (1)蛋白质芯片结果显示,WCX-2芯片在海马中共获得364个蛋白质峰;IMAC-Cu芯片共获得345个蛋白质峰。 (2) 经双向凝胶电泳分离共获得18帧图,图谱蛋白质点分离效果良好,图谱平均匹配率＞80%。PDQuest软件对蛋白质图谱进行定量分析证实,相对分子质量为58.0×103的差异蛋白质为葡萄糖调节蛋白,在损伤后4h、8h和12h表达水平上调;相对分子质量为28.4×103的差异蛋白质为蛋白酶体α亚单位3型,损伤后4h、8h、12h、24h和48h表达水平均上调。 结论 脑损伤可引起海马中蛋白质表达谱发生变化。葡萄糖调节蛋白和蛋白酶体α亚单位3型可能与创伤后脑损伤的发生、发展密切相关。     

深低温脑保护的差异蛋白质组学研究

目的应用差异蛋白质组学技术，研究深低温和常温停循环后大鼠海马组织蛋白质的变化。方法采用深低温停循环模型，取大鼠海马组织，通过双向电泳、分离蛋白，然后通过胶内酶切、生物质谱分析差异的蛋白质点，鉴定出变化的蛋白质。结果通过Image Master软件分析报告发现有差异的Ratio值大于1．5的蛋白考染点14个。通过对这些蛋白考染点进行质谱鉴定，鉴定出28个蛋白质，其中4个为同一种蛋白，实际的蛋白数为24个。它们是细胞骨架蛋白、介导代谢的酶类、参与核酸合成、参与氧化应激反应的蛋白质及未知蛋白。结论鉴定出的差异蛋白质可能与深低温脑保护作用有关，某些蛋白质在低温脑保护中的作用尚未报道。     

王浆蛋白质及其生理药理作用分析

目的 从蛋白质组的角度分析王浆及其生理药理作用,为进一步理解王浆医用价值提供理论基础.方法 提取王浆全蛋白质组分进行双向电泳(2-DE)和质谱鉴定(MALDI-TOF/MS),然后利用蛋白质数据库进行比对.结果 2-DE图谱共检测出152个蛋白点,对其中57个高丰度蛋白进行了MALDI-TOF/MS鉴定,有45个属于王...     

丙戊酸钠对慢性酒中毒模型大鼠学习记忆损害的影响

目的探讨慢性酒中毒对大鼠学习记忆的影响及丙戊酸钠(VPA)的干预效应及其可能机制。方法将56只SD大鼠随机分为酒中毒模型组、VPA干预组、VPA对照组和正常对照组。以乙醇浓度梯度递增式的方式灌胃8周制作慢性酒中毒模型,而第5周始酒中毒模型组腹腔注射生理盐水,VPA干预组于第5～8周腹腔注射VPA,VPA对照组灌注等体积的生理盐水4周后第5～8周给予VPA。8周后每组随机选取7只采用Morris水迷宫和Y迷宫检测大鼠的学习记忆功能,其余7只用Westernblot检测海马脑源性神经营养因子(BDNF)蛋白的表达含量。结果与正常对照组相比,酒中毒模型组大鼠水迷宫的逃避潜伏期显著延长(P0.05),空间探索次数显著减少(P0.05);Y迷宫中2d的错误次数显著增加(P0.01);海马BDNF含量下降(P0.05)。与酒中毒模型组相比,VPA干预组大鼠的行为学成绩均得到改善(P0.01),海马BDNF含量显著增加(P0.01),与正常对照组的差异无统计学意义(P0.05)。VPA对照组与正常对照组各项指标的差异均无统计学意义(P0.05)。结论慢性酒中毒可以导致大鼠学习记忆障碍,而VPA对酒精诱导的学习记忆损害有干预作用,海马BDNF表达增加可能是其作用机制之一。     

蛋白质组学技术鉴定帕金森病模型中的3种新蛋白质

目的 利用蛋白质组学技术对MPP 诱导的PC12细胞帕金森病(PD)模型进行研究,以期发现PD发病机制中的新蛋白.方法 建立MPP 诱导的PC12细胞PD模型并提取细胞总蛋白,荧光差异凝胶电泳(D1GE)进行蛋白样品标记与分离,应用DeCyder软件分析蛋白质差异表达信息,运用MALDI-TOF质谱鉴定差异蛋白质.结果 质谱分析和数据库检索鉴定了3种可能同PD发病相关但在有关PD研究文献中尚未见报道的新蛋白质,他们分别足与线粒体功能相关的蛋白MPPs,具有分子伴侣活性的蛋白NAC和与免疫炎症相关的蛋白gc1qBP.结论 这些新发现蛋白可能与PD的发病机制密切相关.     

五种与癫癎治疗靶点相关蛋白质在颞叶癫癎模型海马组织中的表达

目的寻找颞叶癫大鼠海马组织的差异表达蛋白质,为寻找新的癫治疗靶点和研发新的治疗手段打下基础。方法运用二维电泳和MALD I-TOF-MS技术,对比分析氯化锂-匹罗卡品致大鼠海马组织和正常大鼠海马组织的蛋白质表达谱,对发现的差异表达蛋白质进行分析和鉴定。结果在氯化锂-匹罗卡品致大鼠海马组织中筛选到32个差异表达蛋白质斑点,其中20个在癫组表达下调,12个在癫组表达上调,其中5个蛋白质已被最终鉴定确认,分别为:突触结合蛋白Ⅰ(synaptotagm inⅠ)、神经丝蛋白(neurofilam ents,NF)、热休克蛋白27(heat shock prote in 27,HSP27)、电压依赖性阴离子通道1(voltage-dependent an ion channel prote ins 1,VDAC1)和异柠檬酸脱氢酶(isoc itric dehydrogenase,ICD),其中突触结合蛋白Ⅰ可能为潜在的新治疗靶点。结论颞叶癫大鼠海马组织中存在大量差异表达蛋白质,部分可能为潜在的癫治疗靶点。     

Proteomic analysis of rat prefrontal cortex after chronic valproate treatment

Valproic acid (VPA) is commonly used to treat bipolar disorder (BD), but its therapeutic role has not been clearly elucidated. To gain insights into VPA's mechanism of action, proteomic analysis was used to identify differentially expressed proteins in the rat prefrontal cortex (PFC), a region particularly affected in BD, after 6 weeks of VPA treatment. Proteins from PFCs of control and VPA‐treated rats were separated by 2D‐DIGE and identified by mass spectrometry. Among the 2,826 protein spots resolved, the abundance of 19 proteins was found to be significantly altered in the VPA‐treated group (with the levels of three proteins increasing and 16 decreasing). Seven proteins whose levels were significantly altered after chronic VPA exposure were quantified by Western blot analysis. The 19 identified proteins represent potential new targets for VPA action and should aid in our understanding of the role of VPA in BD. © 2014 Wiley Periodicals, Inc.     

Proteomic Changes in Female Rat Hippocampus Following Exposure to a Terrified Sound Stress

Stress plays a profound role in the onset of affective disorders, including an elevation in risk factors for depression and anxiety. Women are twice as vulnerable to stress as men because of greater sensitivity to a substance produced during times of anxiety. To better define the abnormal proteins implicated in cognitive deficits and other stress-induced dysfunction, female rats were exposed to terrified sound stress, and two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI–TOF MS) were utilized to determine the differential protein expression in the hippocampus in sound-stressed female rats compared with controls. Quantitative differences were found in 44 protein spots which were differentially expressed between the stressed and control groups (fold change of >2; p?<?0.01). Eighteen protein spots were downregulated, and 26 protein spots were upregulated in the stressed group. The seven most differentially expressed proteins were identified and validated as follows: dihydropyrimidinase-related protein 2 (DRP-2), creatine kinase B type, dynamin-1 protein, alpha-internexin, glial fibrillary acidic protein beta, gamma-enolase, and peptidyl-prolyl cis-trans isomerase A. Changes in protein levels were detected in the hippocampus of female rats subjected to terrified sound stress. The findings herein may open new opportunities for further investigations on the modulation induced in the hippocampus by stress at the molecular level, especially with respect to females stress.     

Expression and cellular distribution of multidrug resistance-related proteins in the hippocampus of patients with mesial temporal lobe epilepsy

PURPOSE: This study investigated the cellular distribution of different multidrug resistance (MDR)-related proteins such as P-glycoprotein (P-gp), the multidrug resistance-associated proteins (MRP) 1 and 2, and the major vault protein (MVP) in normal and sclerotic hippocampus of patients with medically refractory mesial temporal lobe epilepsy (MTLE). METHODS: Single- and double-label immunocytochemistry was used on brain sections of control hippocampus and of hippocampus of refractory MTLE patients. RESULTS: In TLE cases with hippocampal sclerosis (HS), all four MDR proteins examined that had low or no expression in control tissue were upregulated, albeit with different cellular distribution patterns. P-gp immunoreactivity (IR) was observed in astrocytes in regions with diffuse reactive gliosis. In 75% of HS cases, strong P-gp IR was detected in blood vessels, with prominent endothelial labeling. Reactive astrocytes displayed low MRP1 IR. However, glial MRP1 expression was noted in glial endfoot processes around blood vessels. Neuronal MRP1 expression was observed in hypertrophic hilar neurons and in a few residual neurons of the CA1 region. Hippocampal MRP2 expression was observed in the large majority of HS cases in blood vessels. Hypertrophic hilar neurons and blood vessels within the sclerotic hippocampus expressed major vault protein (MVP). CONCLUSIONS: These findings indicate that MDR proteins are upregulated in concert in the hippocampus of patients with refractory MTLE, supporting their role in the mechanisms underlying drug resistance. The specific cell-distribution patterns within the sclerotic hippocampus suggest different cellular functions, not necessarily linked only to clinical drug resistance.     

Proteomic analysis of effects of Chinese herbs to calm the liver and suppress hyperactive yang in a rat migraine model***★

BACKGROUND: The expression of ubiquitin and energy-associated protein can provoke migraines. Studies have suggested that expression is closely linked to “hyperactivity of liver-yang theory” in Traditional Chinese Medicine (TCM), as well as the function of periphery sympathetic nerve medulla. OBJECTIVE: To observe proteomic changes in a rat migraine model with regard to hyperactivity of liver-yang when treated with Chinese herbs to calm the liver and suppress hyperactive yang compound. DESIGN, TIME AND SETTING: A randomized controlled study. This study was performed at the laboratory of Institute of Integrated Traditional Chinese and Western Medicine, Institute of Human Reproduction and Stem Cell Engineering and Key Laboratory of Cancer Proteomics of Ministry of Health, Xiangya Hospital Affiliated to Central South University between September 2006 and July 2007. MATERIALS: Thirty, male, healthy, Sprague-Dawley rats, aged eight weeks, were included in the final analysis. Aconite, to calm the liver and suppress hyperactive yang compound, was provided by the Dispensary of Traditional Chinese medicine, Xiangya Hospital, Central South University. A physiological electronic stimulator, type SDQ-1, was provided by Bengbu Practical Institute of Technology. The left trigeminal ganglion was localized and stimulated for 10 minutes, and the rats were orally administered an aconite concoction to establish a rat migraine model with hyperactivity of liver-yang. METHODS: Rats were randomly divided into a normal control group, model group, and TCM treatment group, with 10 rats in each group. The TCM treatment group was orally treated to calm the liver and suppress the hyperactive yang compound once a day for 28 days. In contrast, the model group and normal group were orally administered the same amount of distilled water once a day for 28 days. MAIN OUTCOME MEASURES: The total proteins from adrenal glands of the three groups were separated by two-dimensional gel electrophoresis (2-DE), and 2-DE images were analyzed by PDQuest 7.0 software. Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS) was used to obtain peptide mass fingerprints of the differential proteins. Databases were searched to identify the proteins. RESULTS: A total of 30 rats were included in the final analysis. Reproducible 2-DE patterns from rat adrenal gland of the three groups were obtained. Compared with the normal group, nine proteins were down-regulated and five proteins were up-regulated in the model group; however, these expressions returned to normal, or near normal levels, in the TCM treatment group. A total of eight differentially expressed proteins were identified: glycogen phosphorylase, ATP synthase D chain, isovaleryl-CoA dehydrogenase, ubiquitin, Annexin-3, Annexin-A1, Peroxirdoxin-II, and heat shock protein-27. CONCLUSION: Liver calming and suppression of the hyperactive yang compound may up-regulate expression of proteins related to energy metabolism and the ubiquitin system. Compounds that are used to treat migraines may contribute to protein functions in the peripheral sympathetic nervous system. Key Words: migraine; liver yang hyperactivity; adrenal glands; pacifying liver     

TLR4、MRP8在内侧颞叶癫痫幼大鼠海马中表达的动态变化

目的观察氯化锂-匹罗卡品致痫幼大鼠各期海马中Toll-样受体4(TLR4)、髓样相关蛋白8(MRP8)表达的变化,探讨其是否与内侧颞叶癫痫(MTLE)发生有关。方法 21d SD雄性大鼠90只,随机分对照组(30只)和模型组(60只),腹腔注射氯化锂。17～18h后模型组腹腔注射匹罗卡品诱导癫痫持续状态(SE);对照组予等量生理盐水取代匹罗卡品腹腔注射。按自发发作出现和稳定时间(自发痫性发作在致痫后约3w出现,8w趋稳定),对照组和模型组随机分6个亚组:急性模型组(SE后2h)、潜伏模型组(SE后3w)、慢性自发发作组(SE后8w)及相对应时间点对照组。每亚组动物10只。免疫组化、免疫印迹、RT-PCR技术测定各亚组幼大鼠海马内TLR4、MRP8的表达。结果 TLR4、MRP8在模型组海马内表达明显增多,以CA3、CA1、DG区显著;与对照组相比,差异有显著性(P0.05)。模型亚组内,TLR4、MRP8在急性期和慢性期表达明显增高,而潜伏期无明显表达变化;3组比较差异有显著性(P0.05)。结论大鼠海马内TLR4、MRP8表达增多可能与MTLE发生有关。探讨其机制可能为MTLE的治疗提供新的靶点。     

利用Affymetrix芯片筛选全脑缺血大鼠海马差异表达基因

目的 筛选出全脑缺血大鼠海马差异表达基因.方法 建立大鼠体外循环模型,并将大鼠按随机数字表法分成两组:对照组(n=3),建立体外循环5min后取大鼠海马组织;实验组(n=3),停循环全脑缺血5 min后取大鼠海马组织.采用Affymetrix大鼠全基因组芯片检测两组大鼠海马基因表达的变化,获取差异表达基因.结果 筛选出差异表达基因共55个(上调30个,下调25个),其中28个基因表达差异有统计学意义(P＜0.05).结论 筛选出的全脑缺血大鼠海马差异表达基因对阐明全脑缺血性损伤的分子生物学机制有重要意义.     

Valproate selectively reduces EEG activity in anterior parts of the cortex in patients with idiopathic generalized epilepsy. A low resolution electromagnetic tomography (LORETA) study

PURPOSE: To localize the cortical area where the anticonvulsive drug valproate (VPA) exerts its effect in patients with idiopathic generalized epilepsy (IGE). METHODS: In a prior study we investigated 15 IGE patients in the untreated condition and compared their low resolution electromagnetic tomography (LORETA) results to a normal control group. The investigation of these patients was continued in the present study. All the 15 patients were treated with VPA and were followed by the authors. EEG was recorded after 3 months of VPA treatment in the seizure-free patients. A total of 2min of 19-channels, common reference-recorded, waking-relaxed background activity (without paroxysmal and other, non-stationary elements) was analyzed. "Activity" (current density, amper/meters squared) was given in four frequency bands (delta, theta, alpha, beta). Band-related group differences between the present LORETA results (treated condition) and the prior LORETA results (untreated condition) were computed for all the 2394 voxels by t-tests for interdependent datasets. The statistically significant (p<0.01, uncorrected) differences of activity were projected to real cortical anatomy using the Talairach Brain Atlas. RESULTS: Statistically significant differences between the untreated and treated condition emerged in the delta and theta bands. VPA decreased delta and theta activity in the entire frontal cortex, insula, anterior temporal cortex and hippocampus, and in the anterior part of the parietal cortex. CONCLUSIONS: VPA decreased activity in parts of the cortex that display ictogenic properties and contribute to seizure generation in IGE. Furthermore, the anatomical distribution of the drug effect exactly corresponded to the VPA-related accumulation of neuroprotective proteins reported in experimental papers.     

养血清脑颗粒和丙戊酸联用对戊四氮点燃癫痫大鼠的影响

目的 探讨养血清脑颗粒(YXQNG)联用丙戊酸(VPA)对戊四氮(PTZ)慢性点燃模型大鼠癫痫发作、EEG、认知功能及颞叶、海马T型Ca2+通道蛋白(Cav3.2)表达的影响. 方法 成年雄性SD大鼠40只按随机数字表法分为PTZ组、VPA组、VPA+YXQNG组、NS组,每组10只.前3组大鼠腹腔注射PTZ溶液制作慢性点燃模型,VPA组大鼠在注射PTZ前1 h给予VPA灌胃;VPA+YXQNG组除给予VPA外,注射PTZ前1.5 h给予YXQNG灌胃;NS组腹腔注射生理盐水,每天一次.8周后观察各组大鼠的行为学变化、Y型电迷宫检查大鼠的正确反应率、捕记EEG并应用免疫组化染色检测颞叶和海马Cav3.2的表达. 结果 给药8周后PTZ组大鼠全部达到完全点燃(连续3 d出现Ⅳ级发作或达到Ⅴ级发作),VPA组和VPA+YXQNG组大鼠仅出现0～Ⅱ级发作;Y型电迷宫检查结果显示VPA+YXQNG组大鼠正确反应率高于PTZ组,差异有统计学意义(P＜0.05);EEG结果显示PTZ组大鼠癫痫发作时EEG有明显异常放电,总功率高于用药前,差异有统计学意义(P＜0.05).VPA组、VPA+YXQNG组大鼠用药前后EEG总功率的差值均高于PTZ组,差异有统计学意义(P＜0.05);免疫组化染色结果显示VPA组、VPA+YXQNG组大鼠颞叶和海马Cav3.2表达低于PTZ组,VPA+YXQNG组大鼠颞叶和海马Cav3.2表达低于VPA组,差异均有统计学意义(P＜0.05). 结论 YXQNG和VPA联用能降低癫痫大鼠发作级别、改善认知功能、减少脑部异常放电并降低脑组织Cav3.2水平,有抗癫痫和脑保护作用.

Abstract：

Objective To explore the effet of Yangxue Qingnao granule (YXQNG) on seizures and cognition function of pentylenetetrazole (PTZ)-kindled chronic epileptic rats models, expression of Cav3.2 in the hippocampus and the temporal lobe of these rats, and EEG features of the rats. Methods Forty healthy adult male SD rats were equally divided into 4 groups at random: PTZ group, VPA treatment group, VPA+YXQNG treatment group, normal saline (NS)-control group (n=10). PTZ solution was intraperitoneally injected for 8 weeks to induce the kindling model in the above 3 groups except the NS-control group. VPA by intragastric administration was given to the rats in the VPA treatment group 1 h before PTZ injection; YXQNG and VPA by intragastric administration were given to the rats in the VPA+YXQNG treatment group 1.5 h before PTZ injection. Behavioral changes of the rats were observed 8 weeks after PTZ injection; accuracy rate of response of the rats were examined by electric maze test;EEG was performed; and the expression ofT-type Ca2+ channel protein (Cav3.2) in the temporal lobe and hippocampus was detected by immunohistochemical staining. Results Rats in the PTZ group appeared grade Ⅳ or Ⅴ seizures for 3 consecutive d, and rats in the VPA treatment group, VPA+YXQNG treatment group appeared grade 0-Ⅱ seizures. The accuracy rate of response of the rats in the VPA+YXQNG treatment group was significantly higher than that in the PTZ group (P＜0.05). EEG indicated that paradoxical discharge was noted in rats of PTZ group when seizures appeared, and the total power at the time was obviously higher than that before PTZ injection (P＜0.05). The D-value of total power of EEG in rats of the VPA treatment group and VPA+YXQNG treatment group before and after treatment was significantly higher than that in the PTZ group (P＜0.05). And the level of Cav3.2 in the temporal and hippocampus in rats of the VPA treatment group and VPA+YXQNG treatment group was significantly lower than that in the PTZ group (P＜0.05); as compared with that in the VPA treatment group, the expression of Cav3.2 in the temporal and hippocampus in rats of the VPA+YXQNG treatment group was significantly reduced (P＜0.05). Conclusion The combination use of YXQNG and VPA can decrease the seizure stage, the paradoxical discharge of the brain and the level of Cav3.2 in brain tissue,and improve the cognitive function of the PTZ-kindled rats, indicating that using VAP and YXQNG simultaneously can treat epileptic seizure and protect the neurons.