本文關(guān)鍵詞：石甘散對戊四氮致癇大鼠海馬神經(jīng)元腦源性神經(jīng)營養(yǎng)因子及離子通道的影響　出處：《黑龍江中醫(yī)藥大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 癲癇 石甘散 腦源性神經(jīng)營養(yǎng)因子 離子通道

【摘要】：目的通過觀察戊四氮致癇大鼠經(jīng)石甘散治療前后,實(shí)驗(yàn)大鼠一般狀態(tài)、行為學(xué)、海馬組織病理形態(tài)、離子通道電流幅度及采用免疫組織化學(xué)方法檢測BDNF在海馬內(nèi)表達(dá)的不同,研究石甘散的抗癇的機(jī)制,為石甘散在臨床上用來預(yù)防及治療癲癇,提供必要的理論依據(jù)。方法選取110只健康成年SD大鼠,隨機(jī)選取15只作為空白組,其余大鼠用戊四氮(PTZ)建立大鼠癲癇動(dòng)物模型,造模成功后記錄癲癇大鼠行為學(xué)表現(xiàn),造模成功大鼠隨機(jī)分為模型組、西藥組、石甘散低劑量組、石甘散中劑量組、石甘散高劑量組,西藥組以丙戊酸鈉15.75mg/Kg灌胃治療,中藥組分別予以石甘散0.4g/Kg、0.8g/Kg、1.6g/Kg灌胃,空白組給予蒸餾水3ml/kg灌胃,療程為21天,觀察治療后大鼠行為學(xué)改變、發(fā)作級別變化、體重改變,通過尼氏染色觀察戊四氮致癇大鼠治療后海馬神經(jīng)元病理組織變化,采用免疫組織化學(xué)方法檢測BDNF在海馬內(nèi)表達(dá)的變化,通過全細(xì)胞膜片鉗技術(shù)記錄治療后各組大鼠海馬神經(jīng)元電壓依賴性Na~+通道、K~+通道電流幅度的改變。結(jié)果1.各組大鼠治療前后行為學(xué)改變:(1)體重變化:造模成功后各組大鼠體重與空白組比較均顯著下降(P0.01),治療后,模型組大鼠體質(zhì)量與空白組相比顯著降低(P0.01),其余各組大鼠與空白組比較無差異(P0.05);與治療前相比,各治療組體重增加,石甘散低劑量組具有顯著統(tǒng)計(jì)學(xué)意義(P0.05),西藥組、石甘散中、高劑量組統(tǒng)計(jì)學(xué)意義極顯著(P0.01)。(2)癇性發(fā)作潛伏期(Epileptic seizure latency,ESL)變化:治療前,各組之間癇性發(fā)作潛伏期時(shí)間比較無顯著差異(P0.05)。治療后,模型組癇性發(fā)作潛伏期時(shí)間縮短,其余各組發(fā)作潛伏期時(shí)間均增加,其中,西藥組和石甘散低、中、高劑量組發(fā)作潛伏期時(shí)間顯著增加,統(tǒng)計(jì)學(xué)有其顯著意義(P0.01)。(3)強(qiáng)直性驚厥潛伏期(Tonic convulsion latency,TCL)變化:各組治療后與治療前相比,模型組大鼠強(qiáng)直性驚厥潛伏期顯著降低(P0.05),而其余各組大鼠潛伏期均增加,石甘散低劑量組潛伏期延長,有顯著統(tǒng)計(jì)學(xué)意義(P0.05),西藥組、石甘散中、高劑量組有顯著意義(P0.01)。(4)強(qiáng)直性驚厥發(fā)作率(Tonic seizure rate)變化:治療前后組間比較,模型組強(qiáng)直性驚厥發(fā)作率顯著升高(P0.01),西藥組和石甘散中、高劑量組發(fā)作率比治療前顯著降低(P0.01),石甘散低劑量組與療前比較變化不明顯(P0.05)。2.尼氏染色方法觀察海馬神經(jīng)元病理形態(tài)改變:(1)形態(tài)改變:正常組大鼠CAI區(qū)神經(jīng)元錐體細(xì)胞排列緊密有序,尼氏體深染,形態(tài)規(guī)則,數(shù)量正常;模型組大鼠錐體細(xì)胞形態(tài)皺縮,數(shù)量明顯減少且呈現(xiàn)紊亂排列,尼氏體淺染且有部分溶解;西藥組神經(jīng)元形態(tài)可,細(xì)胞形狀較規(guī)則,部分神經(jīng)元形態(tài)不完整且皺縮,數(shù)量有部分丟失,尼氏體深染;石甘散低劑量組神經(jīng)元錐體細(xì)胞數(shù)量減少,尼氏體淺染溶解,錐體細(xì)胞排列松散且形態(tài)不規(guī)則;石甘散中劑量組錐體細(xì)胞數(shù)量略有減少,形態(tài)略皺縮,尼氏體染色略淺;石甘散高劑量組神經(jīng)元錐體細(xì)胞呈有序排列,形態(tài)尚可,數(shù)量略微減少,尼氏體呈深染。(2)尼氏陽性細(xì)胞數(shù)比較:與空白組比較,模型組和石甘散低劑量組尼氏陽性細(xì)胞數(shù)明顯降低,有顯著統(tǒng)計(jì)學(xué)意義(P0.01);與模型組比較,空白組、西藥組和石甘散中、高劑量組的尼氏陽性細(xì)胞數(shù)均顯著增加(P0.01);各組組間尼氏陽性細(xì)胞數(shù)比較,模型組和石甘散低劑量組細(xì)胞數(shù)明顯減少(P0.01),其余各組細(xì)胞數(shù)與空白組比較差異不大,西藥組和石甘散高劑量組陽性細(xì)胞數(shù)相對較多,且兩者無明顯統(tǒng)計(jì)學(xué)差異(P0.05)。3.治療后PTZ大鼠海馬神經(jīng)元腦源性神經(jīng)營養(yǎng)因子BDNF表達(dá)(1)與空白組比較,模型組、西藥組、石甘散低、中、高劑量組大鼠BDNF的平均灰密度(AGD)均增加,模型組最顯著(P0.01),模型組、西藥組、石甘散低、中、高劑量組大鼠BDNF的平均光密度(AOD)均降低(P0.05),模型組最顯著(P0.01)。(2)與模型組比較,其余各治療組的AGD均降低,西藥組和石甘散中、高劑量組AGD顯著降低(P0.05),石甘散低劑量組無統(tǒng)計(jì)學(xué)意義(P0.05);各組AOD數(shù)值均增加,西藥組和石甘散中、高劑量組AOD增加(P0.05),石甘散低劑量增量無統(tǒng)計(jì)學(xué)意義(P0.05)。(3)組間比較結(jié)果:模型組和石甘散低劑量組的AGD與其余各組比較增加明顯,其中模型組最顯著(P0.01),空白組、西藥組和石甘散中、高劑量組之間AGD無顯著差異(P0.05);模型組和石甘散低劑量組的AOD與其余各組比較降低明顯,其中模型組最顯著(P0.01),空白組、西藥組和石甘散中、高劑量組之間AOD無顯著差異(P0.05)。4.離子通道電流幅度的影響實(shí)驗(yàn)后,與空白組相比,模型組大鼠海馬神經(jīng)元電壓依賴性持續(xù)性Na~+通道電流幅度(INaP)顯著升高(P0.05),與模型組比較,西藥組和石甘散低、中、高劑量組均能降低INaP,差異有統(tǒng)計(jì)學(xué)意義(P0.05),但石甘散低、中、高劑量組和西藥組對電壓依賴性瞬時(shí)性Na~+通道(INat)無顯著改變,統(tǒng)計(jì)學(xué)無意義(P0.05)。與空白組相比,模型組大鼠海馬神經(jīng)元電壓瞬時(shí)外向K~+通道(IA)顯著降低(P0.05),與模型組比較,西藥組和石甘散低、中、高劑量組均能升高降低的IA,差異有統(tǒng)計(jì)學(xué)意義(P0.05),但石甘散低、中、高劑量組和西藥組對延遲整流K~+通道(IK)無顯著改變,統(tǒng)計(jì)學(xué)無顯著差異(P0.05)。結(jié)論1.石甘散低、中、高劑量組和丙戊酸鈉均可以有效增加戊四氮致癇大鼠的體重。2.石甘散低、中、高劑量組與丙戊酸鈉均能明顯延長戊四氮大鼠陣發(fā)性痙攣的潛伏期時(shí)間;石甘散中、高劑量組和丙戊酸鈉能延長大鼠強(qiáng)直性驚厥的潛伏期時(shí)間,并降低大鼠強(qiáng)直性驚厥的發(fā)生率,提示石甘散具有與丙戊酸鈉相似的抗癲癇效應(yīng),且石甘散中、高劑量作用最明顯。3.石甘散中、高劑量組和丙戊酸鈉均可以增加戊四氮模型大鼠尼氏陽性細(xì)胞數(shù)量,改善其病理形態(tài),提示兩者對神經(jīng)元均具有保護(hù)作用。4.石甘散中、高劑量組和丙戊酸鈉均可以降低戊四氮模型大鼠BDNF平均灰密度(Average gray density),增加其平均光密度(Average optical density),說明兩者對大鼠海馬神經(jīng)元具有相似的保護(hù)作用。5.石甘散低、中、高劑量組和丙戊酸鈉均能顯著下調(diào)戊四氮致癇大鼠海馬神經(jīng)元電壓依賴性持續(xù)性Na~+通道電流幅度(INaP),上調(diào)瞬時(shí)外向K~+通道(IA)電流幅度,對電壓依賴性瞬時(shí)性Na~+通道(INat)和延遲整流K~+通道(IK)電流無顯著影響,說明石甘散可能通過對離子通道的調(diào)控發(fā)揮抗癇作用。6.石甘散可能通過增加腦源性神經(jīng)營養(yǎng)因子BDNF含量影響離子通道電流幅度,調(diào)節(jié)靜息膜電位,抑制神經(jīng)突觸傳遞興奮性,發(fā)揮其抗癇作用。
[Abstract]:Objective To observe the e four before and after nitrogen from epileptic rats Gan powder in the treatment of experimental rats, the general state, behavior, hippocampal tissue morphology, ion channel current amplitude and to detect the expression of BDNF in hippocampus by immunohistochemistry method, study of Shi Gan powder anti epilepsy mechanism for Shi Gan powder at clinic for the prevention and treatment of epilepsy, provide the necessary theoretical basis. Methods: 110 healthy adult SD rats, 15 rats were randomly selected as the blank group, the rest rats with e four nitrogen (PTZ) to establish a rat animal model of epilepsy, the successful model of postscript recorded behaviors of rats with epilepsy manifestations, modeling the rats were randomly divided into model group, western medicine group, Shi Gan San Shi Gan powder low dose group, middle dose group, Shi Gan San high dose group, western medicine group with sodium valproate 15.75mg/Kg gavage treatment, traditional Chinese medicine group were given Shi Gan powder 0.4g/Kg, 0.8g/Kg, 1.6g/Kg by gavage, the blank group to Distilled water 3ml/kg orally, 21 days for a course, change the behavior of rats were observed after treatment, onset level changes, weight change, observe the e four nitrogen treatment of epileptic rats after the pathological changes of hippocampal neurons by Nissl staining, changes by immunohistochemical method to detect the expression of BDNF in the sea horse, through the whole cell patch clamp recording after treatment of hippocampal neurons in rats of voltage dependent Na~+ channel, K~+ channel current amplitude changes. The change results before and after treatment of 1. rats in each group: (1) weight change: afterinjecting weight and blank group rats were significantly decreased (P0.01), after treatment, model body mass of rats decreased significantly compared with the control group (P0.01), no difference in other groups with the control group (P0.05); compared with before treatment, the treatment group increased weight, Shi Gan powder low dose group was statistically significant (P0.05), western medicine group, Shi Gan powder in high dose group, significant differences (P0.01). (2) seizure latency (Epileptic seizure, latency, ESL) change: before treatment, the seizure latency time between groups showed no significant difference (P0.05). After treatment, the epileptic model group seizure latency time is shortened, the other groups were increased seizure latency time, among them, the western medicine group and Shi Gan powder, low and high dose group seizure latency time significantly increased, with the statistical significance (P0.01). (3) tetanic convulsion latency (Tonic convulsion, latency, TCL) change: compared each group after treatment with before treatment, tetanic convulsion latency of rats in model group was significantly decreased (P0.05), and the rest of the rats increased latency, Shi Gan powder of low dose group latency was statistically significant (P0.05), western medicine group, Shi Gan powder, are significant in high dose group (P0.0 1). (4) ankylosing convulsion rate (Tonic seizure rate) changes: compared before and after treatment group, model group of ankylosing convulsion rate increased significantly (P0.01), western medicine group and Shi Gan powder in high dose group was significantly lower than before treatment onset (P0.01), low dose of Shi Gan powder group before treatment did not change significantly (P0.05) to observe the pathological changes of hippocampal neurons.2. and Nissl staining method: (1) the morphological changes of neurons in rats: normal group CAI pyramidal cells arranged closely, deep staining, Nissl body shape, the number of normal pyramidal cell shrinkage; morphology of the rats in the model group, the number of decreased and showed a disorganized, Nissl body shallow dyeing and partial dissolution; western medicine group of neurons, the cell shape is regular, some incomplete neurons and the number of shrinkage, partial loss, deep dyeing and Nissl body; Shi Gan powder quantity low dose group of neurons decreased, Nissl body shallow dye dissolved, pyramidal cells arranged in loose and irregular shape; Shi Gan powder quantity in middle dose group, pyramidal cell morphology decreased slightly, slightly wrinkled, Nissl body was slightly shallow; Shi Gan San high dose group of neurons arranged orderly, can form, the number of slightly less, Nissl bodies were anachromasis. (2) comparison of Nissl positive cells: compared with the blank group, model group and Shi Gan powder of low dose group Nissl positive cells decreased significantly, there was statistical significance (P0.01); compared with the model group, blank group, western medicine group and Shi Gan powder, the number of Nissl positive cells the high dose group were significantly increased (P0.01); comparison between each group of Nissl positive cells, model group and Shi Gan powder of low dose group decreased (P0.01), the number of cells in other groups compared with the control group had little difference, the western medicine group and Shi Gan powder relatively high dose group the number of positive cells and, two 鑰呮棤鏄庢樉緇熻瀛﹀樊寮，

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