乳黄片对A型肝性脑病大鼠星形胶质细胞结构与蛋白的影响
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摘要
目的 A型肝性脑病(hepatic encephalopathy,HE)是急性肝功能衰竭并发的大脑功能障碍,该病来势凶险,生存率仅为20%。目前,氨中毒学说被公认为仍处于HE众多发病机制假说的中心地位。通常认为它是通过干扰脑的能量代谢引起HE的发生。近年来,国内外学者认为,氨导致星形胶质细胞结构功能失常,可能是HE发病的主要机理。祖国医学称本病为“肝厥”。根据中医理论与临床经验,导师张赤志教授认为本病病因病机为邪毒直犯肝脏,肝失疏泄,脾失健运,肠道传导失司,腑浊内生,化热生痰,上扰神明;病位在肝、心脑与胃;结合“肝与大肠相通,肝病宜疏通大肠”和“上病下治”治疗原则,立通下解毒法;选用酒制大黄和乳酸菌素组成乳黄片治疗HE,临床和前期动物实验均显示有很好的降氨作用,能明显改善HE症状与体征,提高存活率。为进一步明确乳黄片抗HE的作用位点与机制,本课题从动物和细胞实验两方面研究该药对星形胶质细胞结构和蛋白表达的影响。
方法 (1)200±20gSPF级雄性Wistar大鼠96只,随机分为6组,每组16只:正常对照组(A组)、模型组(B组)、乳果糖组(C组)、乳黄片小中大剂量组(D、E、F组)。大鼠禁食过夜(14h以上),除A组外其余各组采用目前应用最成熟的硫代乙酰胺(TAA)诱导法(TAA250mg/kg灌胃两次,每次间隔24h)复制A型HE动物模型。在TAA首次造模前2h,A、B组每只大鼠灌胃生理盐水1ml/100g体重,其余各组给等体积/重量比的相应药物。以后每8h重复给药一次。自首次TAA造模至实验结束,每隔8h皮下注射3ml 10%葡萄糖、2ml含等量生理盐水与20μmol/lKCl溶液的混合液以防止低血糖。实验30h后行脑电图(EEG)检测,之后处置动物取材。实验过程中,密切观察大鼠行为学改变。(2)眼眶取血,检测血清ALT和血氨浓度。(3)伊文思蓝(EB)渗出法检测血脑屏障(BBB)通透性。透射电镜观察星形胶质细胞在内的BBB超微结构变化。(4)小脑延髓池处抽取脑脊液,检测脑脊液氨浓度。取大脑皮层组织,制成匀浆,高效液相色谱法检测谷氨酰胺含量。取整个脑组织,干湿重法检测脑组织含水量比。(5)免疫组织化学法观察大脑皮层组织星形胶质细胞结构蛋白-胶质纤维酸性蛋白(GFAP)的表达。(6)半定量逆转录-多聚酶链法(RT-PCR)检测大鼠皮层脑组织谷氨酸转运体GLT-1mRNA的表达情况。(7)制备乳黄片脑脊液,作用暴露于5mM浓度氯化铵的星形胶质细胞CRL-2541 48h,MTT法检测其活性,免疫细胞化学法检测GFAP表达情况。
结果 (1)乳黄片可明显改善A型HE大鼠行为异常和EEG变化,显著降低血清ALT与血氨水平(P<0.01),提高存活率;大、中、小剂量组疗效均优于乳果糖组,且以中剂量组最佳;(2)乳黄片能明显降低A型HE大鼠脑组织EB含量(P<0.01),显著改善星形胶质细胞在内的BBB超微结构受损情况,以中剂量组疗效最优;(3)乳黄片可显著降低A型HE大鼠脑脊液氨浓度、脑组织谷氨酰胺含量及脑含水量比(P<0.01),以中剂量组下降最明显;(4)乳黄片能显著上调A型HE大鼠大脑皮层组织星形胶质细胞GFAP的表达,升高
湖北中医学院
博士学位论文
其阳性细胞数和平均光密度(P<0.01),以中剂量上升最显著;(5)乳黄片能
明显上调A型HE大鼠大脑皮层组织星形胶质细胞上谷氨酸转运体
GLT一lmRNA的表达,以中剂量上升最明显;(6)乳黄片脑脊液能显著升高smM
浓度氨环境中星形胶质细胞CRL一2541的吸光度,上调GFAP的表达。
结论(1)高氨是A型HE发病的病因基础之一,其对星形胶质细胞结构和蛋
白的损害在HE的发病机理中占有重要作用。其中,氨及其代谢产物一谷氨
酞胺的过量堆积可引起星形胶质细胞结构损害和GFAP蛋白表达缺失,导致
血脑屏障完整性受损,通透性增加和血管源性脑水肿的发生;继发的谷氨
酸转运体蛋白(如GLT一1)基因表达缺失,可导致谷氨酸大量积于突触间隙,
引起神经元毒性和脑水肿的发生。(2)乳黄片有良好防治A型HE的作用,可
能是通过以下途径实现的:保肝护肝,降低氨(血和脑脊液氨)与谷氨酞胺
浓度,进入血脑屏障,改善星形胶质细胞结构受损与蛋白表达缺失情况。
(3)乳黄片抗HE的作用位点可能为星形胶质细胞。
关键词:肝性脑病/A型@乳黄片星形胶质细胞实验研究
Objective Hepatic encephalopathy (HE) of type A is a kind of encephalopathies associated with acute liver function failure. It is dangerous and the survival rate of it is only 20%. At present, ammonia intoxication is still in the center of the pathogeneses of HE. Usually, it was thought that ammonia caused HE by interacting brain energy. Recently, the domestic and abroad scholars think the main pathogenesis of HE maybe the disorders of astroglia structure and functions caused by ammonia. In Traditional Chinese Medicine (TCM), it was called "GanJue" . Based on TCM theories and clinical experiences, my tutor -professor Zhang ChiZhi think that the pathogenesis of Ganjue is the result of heat and phlegm troubling mind which caused by the disorders of liver, speel and intestine. The sick position of it is liver, heart, brain and stomach. According to TCM therapeutic principle of "liver and large intestine are collected with each other, diseases of liver are fit to clean large intestine " and "be ill upper, treat down" , professor Zhang uses wined Rhubarb and Lactohacillin to make RuHuang Pill. Clinic and animal studies showed it had good effects on the treatment of HE. It could decline ammonia concentration, obviously ameliorative the symptoms and signs of HE, increase survive rate. The study is to observe the effects of RuHuang pill on the structure and protein expressions of astroglia, to reveal the function point and mechanism of it. Methods(l)96 male Wistar rats weighted 200±20g of SPF grade were randomly divided into six groups: Normal control group (group A), model group (group B), lactulose group (group C), RuHuang Pill group of small dose (group D), RuHuang Pill group of middle dose (group E), RuHuang Pill group of large dose (group F). After fasted overnight for over 14h, all rats except group A were induced to be model of HE of type A by administration with TAA 250mg/ kg (twice, per 24 h once). Before two hours of administration with TAA, each rat in group A and group B were given normal saline 1ml/100g weight, and the left were given the same volume/weight medicine respectively, then redone every 8h. From the first administration with TAA to the end of experiment, every rat was injected mixture solution contained 10% glucose 3ml, saline 1ml, and 20umol/l KC1 lml every 8h to avoid hypoglucose. At 30h of the experiment, electroencephalogram (EEG) examination was done in each group, and then all rats were sacrificed. During the experiment, the behaviors of all rats were observed closely. (2) Blood from eye socket was used to examine the concentrations of serum ALT and blood ammonia. (3) The permeability of blood-brain barrier (BBB) was detected by Evans Blue (EB) leakage method, and the ultrastruncture of BBB, which contained astroglia, was observed under electron microscopy. (4) The cerebrospinal fluid collected from cerebellommedullary cistern was used to determine the concentrations of
ammonia. Cerebral cortexes homogenized were used to examine the contents of glutamine by high performance liquid chromatography. Whole brain tissues were used to determine the percentages of brain water content by dry-wet method. (5)The expressions of glial fibrillary acidic protein (GFAP) in cerebral cortex, which was an important structure protein of astroglia, were observed via immunocytochemistry. (6)The expressions of GLT-1mRNA in cerebral cortex, which was a kind of astroglia glutamate transporters, were detected by reverse transcription-coupled polymerase chain reaction (RT-PCR). (7) Cerebrospinal fluid of RuHuang Pill was prepared to effect astroglia CRL-2541 exposed to 5mM concentration of NH4C1. After 48h treatment, the MTT tests were performed to observe the effect of drug on the rate of CRL-2541 survival, immunocytochemistries were performed to examine the expressions of GFAP. Result (1) RuHuang pill could obviously ameliorative the disorders of behaviors and EEGs of rats with HE of type A, decline the concentrations of serum ALT and blood ammonia(P<0.01), increase survive rates. Three dose groups of RuHuang P
方法 (1)200±20gSPF级雄性Wistar大鼠96只,随机分为6组,每组16只:正常对照组(A组)、模型组(B组)、乳果糖组(C组)、乳黄片小中大剂量组(D、E、F组)。大鼠禁食过夜(14h以上),除A组外其余各组采用目前应用最成熟的硫代乙酰胺(TAA)诱导法(TAA250mg/kg灌胃两次,每次间隔24h)复制A型HE动物模型。在TAA首次造模前2h,A、B组每只大鼠灌胃生理盐水1ml/100g体重,其余各组给等体积/重量比的相应药物。以后每8h重复给药一次。自首次TAA造模至实验结束,每隔8h皮下注射3ml 10%葡萄糖、2ml含等量生理盐水与20μmol/lKCl溶液的混合液以防止低血糖。实验30h后行脑电图(EEG)检测,之后处置动物取材。实验过程中,密切观察大鼠行为学改变。(2)眼眶取血,检测血清ALT和血氨浓度。(3)伊文思蓝(EB)渗出法检测血脑屏障(BBB)通透性。透射电镜观察星形胶质细胞在内的BBB超微结构变化。(4)小脑延髓池处抽取脑脊液,检测脑脊液氨浓度。取大脑皮层组织,制成匀浆,高效液相色谱法检测谷氨酰胺含量。取整个脑组织,干湿重法检测脑组织含水量比。(5)免疫组织化学法观察大脑皮层组织星形胶质细胞结构蛋白-胶质纤维酸性蛋白(GFAP)的表达。(6)半定量逆转录-多聚酶链法(RT-PCR)检测大鼠皮层脑组织谷氨酸转运体GLT-1mRNA的表达情况。(7)制备乳黄片脑脊液,作用暴露于5mM浓度氯化铵的星形胶质细胞CRL-2541 48h,MTT法检测其活性,免疫细胞化学法检测GFAP表达情况。
结果 (1)乳黄片可明显改善A型HE大鼠行为异常和EEG变化,显著降低血清ALT与血氨水平(P<0.01),提高存活率;大、中、小剂量组疗效均优于乳果糖组,且以中剂量组最佳;(2)乳黄片能明显降低A型HE大鼠脑组织EB含量(P<0.01),显著改善星形胶质细胞在内的BBB超微结构受损情况,以中剂量组疗效最优;(3)乳黄片可显著降低A型HE大鼠脑脊液氨浓度、脑组织谷氨酰胺含量及脑含水量比(P<0.01),以中剂量组下降最明显;(4)乳黄片能显著上调A型HE大鼠大脑皮层组织星形胶质细胞GFAP的表达,升高
湖北中医学院
博士学位论文
其阳性细胞数和平均光密度(P<0.01),以中剂量上升最显著;(5)乳黄片能
明显上调A型HE大鼠大脑皮层组织星形胶质细胞上谷氨酸转运体
GLT一lmRNA的表达,以中剂量上升最明显;(6)乳黄片脑脊液能显著升高smM
浓度氨环境中星形胶质细胞CRL一2541的吸光度,上调GFAP的表达。
结论(1)高氨是A型HE发病的病因基础之一,其对星形胶质细胞结构和蛋
白的损害在HE的发病机理中占有重要作用。其中,氨及其代谢产物一谷氨
酞胺的过量堆积可引起星形胶质细胞结构损害和GFAP蛋白表达缺失,导致
血脑屏障完整性受损,通透性增加和血管源性脑水肿的发生;继发的谷氨
酸转运体蛋白(如GLT一1)基因表达缺失,可导致谷氨酸大量积于突触间隙,
引起神经元毒性和脑水肿的发生。(2)乳黄片有良好防治A型HE的作用,可
能是通过以下途径实现的:保肝护肝,降低氨(血和脑脊液氨)与谷氨酞胺
浓度,进入血脑屏障,改善星形胶质细胞结构受损与蛋白表达缺失情况。
(3)乳黄片抗HE的作用位点可能为星形胶质细胞。
关键词:肝性脑病/A型@乳黄片星形胶质细胞实验研究
Objective Hepatic encephalopathy (HE) of type A is a kind of encephalopathies associated with acute liver function failure. It is dangerous and the survival rate of it is only 20%. At present, ammonia intoxication is still in the center of the pathogeneses of HE. Usually, it was thought that ammonia caused HE by interacting brain energy. Recently, the domestic and abroad scholars think the main pathogenesis of HE maybe the disorders of astroglia structure and functions caused by ammonia. In Traditional Chinese Medicine (TCM), it was called "GanJue" . Based on TCM theories and clinical experiences, my tutor -professor Zhang ChiZhi think that the pathogenesis of Ganjue is the result of heat and phlegm troubling mind which caused by the disorders of liver, speel and intestine. The sick position of it is liver, heart, brain and stomach. According to TCM therapeutic principle of "liver and large intestine are collected with each other, diseases of liver are fit to clean large intestine " and "be ill upper, treat down" , professor Zhang uses wined Rhubarb and Lactohacillin to make RuHuang Pill. Clinic and animal studies showed it had good effects on the treatment of HE. It could decline ammonia concentration, obviously ameliorative the symptoms and signs of HE, increase survive rate. The study is to observe the effects of RuHuang pill on the structure and protein expressions of astroglia, to reveal the function point and mechanism of it. Methods(l)96 male Wistar rats weighted 200±20g of SPF grade were randomly divided into six groups: Normal control group (group A), model group (group B), lactulose group (group C), RuHuang Pill group of small dose (group D), RuHuang Pill group of middle dose (group E), RuHuang Pill group of large dose (group F). After fasted overnight for over 14h, all rats except group A were induced to be model of HE of type A by administration with TAA 250mg/ kg (twice, per 24 h once). Before two hours of administration with TAA, each rat in group A and group B were given normal saline 1ml/100g weight, and the left were given the same volume/weight medicine respectively, then redone every 8h. From the first administration with TAA to the end of experiment, every rat was injected mixture solution contained 10% glucose 3ml, saline 1ml, and 20umol/l KC1 lml every 8h to avoid hypoglucose. At 30h of the experiment, electroencephalogram (EEG) examination was done in each group, and then all rats were sacrificed. During the experiment, the behaviors of all rats were observed closely. (2) Blood from eye socket was used to examine the concentrations of serum ALT and blood ammonia. (3) The permeability of blood-brain barrier (BBB) was detected by Evans Blue (EB) leakage method, and the ultrastruncture of BBB, which contained astroglia, was observed under electron microscopy. (4) The cerebrospinal fluid collected from cerebellommedullary cistern was used to determine the concentrations of
ammonia. Cerebral cortexes homogenized were used to examine the contents of glutamine by high performance liquid chromatography. Whole brain tissues were used to determine the percentages of brain water content by dry-wet method. (5)The expressions of glial fibrillary acidic protein (GFAP) in cerebral cortex, which was an important structure protein of astroglia, were observed via immunocytochemistry. (6)The expressions of GLT-1mRNA in cerebral cortex, which was a kind of astroglia glutamate transporters, were detected by reverse transcription-coupled polymerase chain reaction (RT-PCR). (7) Cerebrospinal fluid of RuHuang Pill was prepared to effect astroglia CRL-2541 exposed to 5mM concentration of NH4C1. After 48h treatment, the MTT tests were performed to observe the effect of drug on the rate of CRL-2541 survival, immunocytochemistries were performed to examine the expressions of GFAP. Result (1) RuHuang pill could obviously ameliorative the disorders of behaviors and EEGs of rats with HE of type A, decline the concentrations of serum ALT and blood ammonia(P<0.01), increase survive rates. Three dose groups of RuHuang P
引文
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31 王浴生,邓文龙,薛春生.中药药理与应用.北京:人民卫生出版社,2000:70-74
32 陈德昌,李红红,高春芳,等.大黄对烫伤大鼠肝脏肿瘤坏死因子受体基因表达的影响.中国中西医结合急救杂志,2000:7(1):5-8
33 过建春,石伟珍,等.生大黄为主治疗重型肝炎疗效观察.中西医结合肝病杂志,1997:7(2):118-120
34 焦东海.大黄在急救医学中的应用概述.中国中西医结合急救杂志,2000:7(1):3-4
35 廖树琪,毛德文.大黄煎剂保留灌肠治疗重型肝炎40例总结.湖南中医杂志,2002;18(4):9-10
36 于秋,张丽香,等.大黄食醋液保留灌肠治疗肝性脑病的临床观察.实用中西医结合杂志,1998:11(1):72-73
37 江文君,等.泡制大黄加酒与否对作用与成份影响的研究,鉴定会资料“熟大黄泡制工艺研究”专集.1984:65
38 陈馥馨,高小山,等.大黄十种不同煎煮法的部分药效学比较研究.中成药,1990:12(7):2～4
39 熊德鑫主编.现代微生学[M].北京:中国科技出版社,2000:20-63
40 那淑敏,李立波.嗜酸乳杆菌发酵代谢产物分析.中国微生态学杂志,1999:11(5):266
41 张凤莲,付惠玲,朱智玲,等.乳酸菌素治疗消化性溃疡的临床分析.中国微生态学杂志,2003:15(2):105
42 林敏西,谢建萍,刘安国,等.乳酸菌素治疗HBV感染与肠源性内毒素血症的临床研究.中国微生态学杂志,2000:12(5):283
43 郭恩建,戴建红,林敏西,等.体外乳酸菌素促双歧杆菌生长实验初步观察.中国微生态学杂志,2000:12(5):266
44 张民庆,张名伟,唐德才.现代临床中药学.上海:上海中医药大学出版社.2002:174
45 范志明,秦靖,高金凤,等.微生态调节治疗对肝硬化病人内毒素及肿瘤坏死因子影响.中华实验和临床病毒学杂志,1994:8(4):361-363
2 张赤志,陈军梅,魏玮.酒制大黄、乳酸菌素及复方乳黄制剂对急性肝性脑病大鼠的防治研究.中西医结合肝病杂志,2004:14(6):347-350
3 田建锋,李晓玉,刘建华,等.培菲康对硫代乙酰胺诱导的大鼠肝性脑病的保护作用.中国药理学通报,2001;17(1):86-87
4 芮长江,江净.肝性脑病脑电图的临床应用.中国基层医药,2003:10(9):909
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6 赵明耀,刘守彦,汤宁,等.用小白鼠复制肝性脑病的氨中毒动物模型.河南医科大学学报,1996;31(4):93-94
7 张宗明,吴才宏,周培爱,等.肝性脑病大鼠海马CA1区锥体细胞氨基酸激活电流改变.中华医学杂志,1997;77(6):440
8 张宗明,裘法祖,陈孝平.肝性脑病兔门静脉血沁脏血及脑脊液内血管活性肠肽和生长抑素改变.中华实验外科杂志,1994:11(5):279
9 Adrianna Michalak, Roger F Butterworth. Selective loss of binding sites for the glutamate receptor ligands [3H] Kainate and (s)-[3H] 5-Fluorowillardiine in the brains of rats with acute liver failure. Hepatology, 1997; 25(3): 631-632
10 张宗明,裘法祖,陈孝平.肝性脑病大白鼠脑内外周型苯二氮卓受体的研究.中华外科杂志,1994:32(1):37-38
11 熊益群,周大桥,李航森,等.实验性大鼠肝性脑病动物模型的研究.中国实验动物学杂志,2002:12(6):342
12 Rahman TM, Selden AC, Hodgson HJ. A novel model of acetaminophen-induced acute hepatic failure in rabbits. J Surg Res. 2002; 106(2): 264-272
13 Matkowskyj KA, Marrero JA, Carroll RE, et al. Azoxymethane-induced fulminant hepatic failure in C57BL/6J mice: characterizaion of a new animal model. Am J Physiol, 1999; 277(2pt1): 6455-462
14 Chu CJ, Lee FY, Wang SS, et al. Establishment of an animal model of hepatic encephalopathy. ZhongHuaYiXue ZaZhi (TaiPei), 2000; 63(4): 263-269
15 任大宾,韩德五,赵元昌.急性肝衰竭时肠源性内毒素血症对肝脏能量代谢的影响.中国病理生量杂志,2001:17(9):890-892
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17 Margeli AP, Papadimitriou L, Ninos S, et al. Hepatic stimulator substance administration ameliorats liver regeneration in an animal model of fulminant hepatic failure and encephalopathy. Liver Int, 2003; 23(3): 171-178
18 李瑞军,唐晓山,杨昭徐.中药提取物水苏糖对临床/亚临床肝性脑病的预防作用.中国新药杂志,2001:10(6):429-430
19 Kofi NW Oppong, Kim Bartlett, Christopher O, et al. Synaptosomal glutamate transport in thioacetamide-induced hepatic encephalopathy in the rat. Hepatology, 1995; 22(2): 553-554
20 Christof Zimmermann, Peter Ferenci, Christian Pifl, et al. Hepatic encephalopathy in thioacetamide-induced acute liver failure in rats: characterization of an improved model and study of amino acid-ergic neurotransmission. Hepatology, 1989; 9(4): 594-601
21 许瑞龄,尹镭,陈贤明,等.暴发性肝衰竭血脑屏障通透性改变对肝性脑病发生发展的影响.中国病理生理杂志,1994:10(2):160-161
22 刘厚钰,石虹.肝性脑病(一).胃肠病学,2002;7(6):383
23 戚仁锋.诊断学[M].北京:人民卫生出版社,1996:415
24 Peter C Hayes.肝性脑病.国际内科双语杂志,2003:3(2):86-88
25 Kramer L, Tribl B, Gendo A, et al. Partial pressure of ammonia versus ammonia in hepatic encephalopathy. Hepatology, 2000; 31: 30-34
26 周镇先.血氨水平与肝性脑病关系的再认识.江苏医药杂志,2003;29(7):560
27 刘厚珏,石虹.肝性脑病(二).胃肠病学,2003:8(1):61
28 同 24:1014
29 焦东海,章启尧.精制大黄片治疗急性病毒性肝炎的研究.中医杂志,1990:(9):35
30 阴健.中药现代研究与临床应用.北京:学苑出版社,1993:50
31 王浴生,邓文龙,薛春生.中药药理与应用.北京:人民卫生出版社,2000:70-74
32 陈德昌,李红红,高春芳,等.大黄对烫伤大鼠肝脏肿瘤坏死因子受体基因表达的影响.中国中西医结合急救杂志,2000:7(1):5-8
33 过建春,石伟珍,等.生大黄为主治疗重型肝炎疗效观察.中西医结合肝病杂志,1997:7(2):118-120
34 焦东海.大黄在急救医学中的应用概述.中国中西医结合急救杂志,2000:7(1):3-4
35 廖树琪,毛德文.大黄煎剂保留灌肠治疗重型肝炎40例总结.湖南中医杂志,2002;18(4):9-10
36 于秋,张丽香,等.大黄食醋液保留灌肠治疗肝性脑病的临床观察.实用中西医结合杂志,1998:11(1):72-73
37 江文君,等.泡制大黄加酒与否对作用与成份影响的研究,鉴定会资料“熟大黄泡制工艺研究”专集.1984:65
38 陈馥馨,高小山,等.大黄十种不同煎煮法的部分药效学比较研究.中成药,1990:12(7):2～4
39 熊德鑫主编.现代微生学[M].北京:中国科技出版社,2000:20-63
40 那淑敏,李立波.嗜酸乳杆菌发酵代谢产物分析.中国微生态学杂志,1999:11(5):266
41 张凤莲,付惠玲,朱智玲,等.乳酸菌素治疗消化性溃疡的临床分析.中国微生态学杂志,2003:15(2):105
42 林敏西,谢建萍,刘安国,等.乳酸菌素治疗HBV感染与肠源性内毒素血症的临床研究.中国微生态学杂志,2000:12(5):283
43 郭恩建,戴建红,林敏西,等.体外乳酸菌素促双歧杆菌生长实验初步观察.中国微生态学杂志,2000:12(5):266
44 张民庆,张名伟,唐德才.现代临床中药学.上海:上海中医药大学出版社.2002:174
45 范志明,秦靖,高金凤,等.微生态调节治疗对肝硬化病人内毒素及肿瘤坏死因子影响.中华实验和临床病毒学杂志,1994:8(4):361-363