苦参素对实验性自身免疫性脑脊髓炎大鼠防治机制探讨

发布时间：2018-05-08 09:33

  本文选题：苦参素 + 实验性自身免疫性脑脊髓炎　； 参考：《郑州大学》2014年硕士论文

【摘要】：目的： 观察苦参素（Matrine，MAT）对多发性硬化症（multiple sclerosis，MS）动物模型实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis，EAE)大鼠临床症状、组织病理学的影响，分析大鼠发病过程中血脑屏障体系(blood-brain barrier，BBB)相关因子如IV型胶原蛋白（Collagen IV），闭锁小带蛋白-1（Zonula occludens-1， ZO-1），基质金属蛋白酶-2（matrixmetalloproteinase-2，MMP-2），基质金属蛋白酶-9（matrix metalloproteinase-9，MMP-9），基质金属蛋白酶抑制剂-1（tissue inhibitors of metalloproteinase-1，TIMP-1），基质金属蛋白酶抑制剂-2（tissue inhibitors of metalloproteinase-2，TIMP-2）；谷氨酸兴奋系统相关因子谷氨酸（glutamate，Glu），γ-氨基丁酸（γ-aminobutyric acid，GABA），谷氨酸转运体-1（glutamate transport-1，GLT-1），谷氨酸/天冬氨酸转运体（glutamate/aspartate transporter，GLAST），N-甲基-D-天冬氨酸受体（N-methyl-d-aspartic acid receptor，NMDAR），α-氨基羟甲基恶唑丙酸受体(α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor，AMPAR)；轴突生长抑制因子-A（Nogo-A）及轴突生长抑制因子受体（Nogoreceptor，NgR）表达的变化，探究苦参素对EAE的防治效果及作用机制，旨在为多发性硬化症提供一种安全、有效、经济的治疗药物。 方法： 1建立Wistar大鼠EAE模型：在无菌条件下制备豚鼠全脊髓匀浆（guinea pigspinal cord homogenate，GPSCH），与等体积含6mg/ml卡介苗的完全弗氏佐剂（complete freund adjuvant，CFA）制备稳定油包水型抗原乳剂作为抗原，给予Wistar雌性大鼠足垫皮内注射诱导EAE模型。自免疫日起，详细观察并记录大鼠体重变化、饮食情况、活动能力变化情况、临床症状及神经功能评分。 2动物分组及给药：将40只6～8周龄免疫后的Wistar雌性大鼠采用随机数字表法分为四组：EAE模型组（Vehicle）、MAT低剂量组（MAT-L）、MAT高剂量组（MAT-H）、地塞米松组（DEX），每组10只。同时有10只健康大鼠作为健康对照组(Naive)。从免疫当日起，五组大鼠分别给予不同干预措施，其中MAT低剂量组每日给予1次6.7ml/kg(150mg/kg)苦参素注射液腹腔注射治疗，MAT高剂量组每日给予1次6.7ml/kg(200mg/kg)苦参素注射液腹腔注射治疗，DEX组每日给予1次6.7ml/kg(1mg/kg)地塞米松注射液腹腔注射治疗，正常对照组和模型组采用等量生理盐水替代治疗，，五组大鼠均连续干预16天。 3动物标本收集：在免疫第17天，使用10%水合氯醛麻醉后，眼眶后静脉丛取血3ml，制血清备用；预冷生理盐水心脏灌流完全后，留取大鼠脑与脊髓标本，部分用于制作石蜡切片，其余立即投入液氮留存备用。 4相关指标检测：苏木素-伊红（HE）和变色酸-2R-亮绿（C-2R-BG）染色观察炎性细胞浸润及脱髓鞘情况，免疫组织化学法分析MMP-2、TIMP-2、NMDAR、AMPAR、Nogo-A表达，酶联免疫吸附法（ELISA法）检测MMP-9、TIMP-1、Glu、GABA水平，逆转录聚合酶链反应法（RT-PCR法）分析CollagenIV、ZO-1、GLT-1、GLAST、NgR含量。 结果： 1发病率：每组10只动物中，正常对照组大鼠未观察到发病情况，模型组有8只发病，MAT-L组有6只发病，MAT-H组有5只发病，DEX组有4只发病。Fisher确切概率分析结果示：正常对照组发病率显著低于模型组(p 0.05)；MAT-L组、MAT-H组、DEX组大鼠发病率均低于模型组，但是差异不具有统计学意义(p0.05)。此外三个药物干预组间亦不存在显著性差异(p0.05)。 2体重变化情况：正常对照组大鼠自免疫第2天起平均体重稳步上升，其余四组平均体重总体上均呈现上升后下降趋势。两个MAT干预组体重降低幅度显著低于模型组(p 0.01)，亦低于地塞米松组(p 0.01)。且与MAT-L组相比，MAT-H组大鼠平均体重变化较小，差异具有统计学意义(p 0.05)。 3神经功能学评分：本研究中正常对照组平均神经评分一直保持为0。免疫后第17天，模型组大鼠平均神经功能学评分显著高于三个药物干预组(p 0.05)。三个药物干预组间平均神经功能评分无显著性差异(p0.05)。 4组织病理学情况：模型组平均炎症浸润评分和髓鞘脱失评分较正常对照组明显升高，差异具有显著性意义(p 0.01)。MAT-L组平均炎症浸润评分和髓鞘脱失评分显著低于模型组(p 0.01)。与MAT-L组相比，MAT-H组平均炎症浸润评分和髓鞘脱失评分进一步降低(p 0.05)。与MAT-H组相比，DEX组平均炎症浸润评分显著性降低(p 0.01)，髓鞘脱失评分无显著性差异(p0.05)。 5脊髓中Collagen IV、ZO-1mRNA的表达：与正常对照组相比，模型组Collagen IV，ZO-1mRNA含量显著性降低(p 0.01)。两个MAT干预组CollagenIV，ZO-1mRNA表达均较模型组明显升高(p 0.01)，其中MAT-H组优于MAT-L组(p 0.01)。MAT-H组、DEX组在Collagen IV，ZO-1mRNA表达方面均无显著性差异(p0.05)。 6血清中MMP-9、TIMP-1水平：与正常对照组相比，模型组大鼠血清中MMP-9含量明显升高(p 0.01)。两个MAT干预组MMP-9水平均较模型组显著性降低(p 0.01)，其中MAT-H组降低幅度更为明显(p 0.01)。DEX组在抑制MMP-9病理性升高方面优于MAT-H组，差异存在统计学意义(p 0.01)。模型组较正常对照组血清中TIMP-1含量明显降低(p 0.01)。DEX组TIMP-1水平均较模型组显著性增高(p 0.01)；MAT升高EAE大鼠血清TIMP-1的能力存在剂量依赖性(p 0.01)。与MAT-H组相比，DEX组TIMP-1水平较低，差异具有统计学意义(p 0.01)。 7脊髓中MMP-2、TIMP-2含量：模型组较正常对照组脊髓中MMP-2含量明显升高(p 0.01)。两个MAT干预组MMP-2水平均较模型组显著性降低(p 0.01)，其中MAT-H组降低幅度更为明显(p 0.05)。DEX组MMP-2表达低于MAT-H组，但差异不具有统计学意义(p0.05)。与正常对照组相比，模型组大鼠血清中TIMP-2含量明显减低(p 0.01)。三个药物组TIMP-2水平均较模型组显著性增高(p 0.05)。MAT升高EAE大鼠血清TIMP-1的能力存在剂量依赖性(p 0.01)。MAT-H组与DEX组在TIMP-2含量方面无显著性差异(p0.05)。 8大脑皮质Glu、GABA含量：与正常对照组相比，模型组大鼠脑皮质Glu含量明显升高(p 0.01)。MAT-L组Glu含量较模型组低，但是差异不具有统计学意义(p0.05)。MAT-H组与DEX组Glu含量均比模型组低(p 0.05)，但是两者间无显著性差别(p0.05)。模型组大鼠脑皮质GABA含量比正常对照组显著性降低(p 0.01)。MAT升高EAE脑皮质GABA的能力存在剂量依赖性(p 0.05)。与DEX组相比，MAT-L组GABA水平较低，差异具有统计学意义(p 0.05)。MAT-H组与DEX组在脑皮质GABA含量方面不存在显著性差异(p0.05)。 9大脑皮质GLT-1、GLAST mRNA表达：与正常对照组相比，模型组GLT-1，GLAST mRNA含量显著降低(p 0.01)；三个药物干预组GLT-1mRNA表达均较模型组显著性升高(p 0.01)，但组间差异不具有显著性(p0.05)。MAT-L组和MAT-H组GLAST mRNA表达均较模型组明显升高(p 0.01)，其中MAT-H组更高，但差异不具有统计学意义(p0.05)。高剂量MAT在提高GLAST mRNA含量方面优于DEX (p 0.05)。 10大脑皮质NMDAR、AMPAR表达：模型组较正常对照组大脑皮质中NMDAR、AMPAR含量明显升高(p 0.01)。两个MAT干预组NMDAR、AMPAR水平均较模型组显著性降低(p 0.05)，其中MAT-H组降低幅度更为明显(p 0.01)。MAT-H组AMPAR表达低于DEX组，差异具有统计学意义(p 0.05)；但是两组在NMDAR表达方面无统计学差异(p0.05)。 11脊髓中Nogo-A蛋白、NgR mRNA表达：模型组较正常对照组脊髓中Nogo-A蛋白，NgR mRNA含量明显升高(p 0.01)。两个MAT干预组Nogo-A蛋白，NgR mRNA水平均较模型组显著性降低(p 0.01)，其中MAT-H组降低幅度更为明显(p 0.01)。MAT-H组Nogo-A蛋白，NgR mRNA表达高于DEX组，差异具有统计学意义(p 0.01)。 结论： MAT对Wistar大鼠EAE具有良好的防治作用，可能是通过上调机体血脑屏障基质膜、紧密连接蛋白，维持体内MMPs与TIMPs的平衡；下调谷氨酸及其受体含量，提高其天然抑制剂GABA和谷氨酸转运体的表达；抑制Nogo-A及受体NgR水平，发挥保护大鼠血脑屏障、抑制谷氨酸系统过度激活、下调Nogo-A/NgR轴索抑制通路兴奋性作用，从而起到对EAE大鼠的防治效果。
[Abstract]:Purpose :

To observe the effects of Matrine ( MAT ) on the clinical symptoms and histopathological changes in rats with multiple sclerosis ( MS ) models and to analyze the effects of matrix metalloproteinase - 2 ( MMP - 2 ) , matrix metalloproteinase - 9 ( MMP - 9 ) , matrix metalloproteinase - 1 ( MMP - 2 ) , matrix metalloproteinase - 9 ( MMP - 9 ) , matrix metalloproteinase - 1 ( MMP - 2 ) , matrix metalloproteinase - 9 ( TIMP - 1 ) , matrix metalloproteinase - 2 ( tissue inhibitors of metalloproteinase - 2 , TIMP - 2 ) .
glutamate ( glutamate , Glu ) , gamma - aminobutyric acid ( GABA ) , glutamate transporter - 1 ( glutamate transport - 1 , GLT - 1 ) , glutamate / aspartate transporter ( GLAST ) , N - methyl - D - aspartate receptor ( N - methyl - d - glutamic acid receptor , NMDAR ) , 伪 - amino - methyloxacillin - propionic acid receptor ( AMPAR ) ;
Objective To explore the effect and mechanism of matrine on the prevention and treatment of multiple sclerosis and to provide a safe , effective and economical drug for multiple sclerosis .

Method :

1 Wistar rat model was established : guinea pig whole spinal cord homogenate ( GPSCH ) was prepared under aseptic conditions . Complete freund adjuvant ( CFA ) was prepared by complete freund adjuvant ( CFA ) .

Two groups of rats were randomly divided into four groups : Vehicle , MAT - L , MAT - H and DEX . At the same time , 10 healthy rats were injected intraperitoneally with 6 . 7ml / kg ( 150 mg / kg ) of Matrine injection .

3 Animal specimens collected : After 17 days of immunization , after anesthesia with 10 % chloral hydrate , 3 ml of blood were taken from the posterior vein plexus of the orbit , and the serum was prepared for standby ;
After the pre - cold normal saline heart perfusion was complete , the rat brain and spinal cord specimens were removed , partially used to make paraffin sections , and the rest was put into liquid nitrogen for standby immediately .

The expression of MMP - 2 , TIMP - 2 , NMDAR , AMPAR , Nogo - A and enzyme - linked immunosorbent assay ( ELISA ) were used to detect MMP - 9 , TIMP - 1 , Glu , GABA levels and reverse transcription polymerase chain reaction ( RT - PCR ) .

Results :

1 Incidence rate : In 10 animals in each group , the incidence was not observed in the normal control group , 8 in the model group , 6 in the MAT - L group , 5 in the MAT - H group , 4 in the DEX group , and the Fisher exact probability analysis showed that the incidence of the normal control group was significantly lower than that of the model group ( p 0.05 ) ;
The morbidity of MAT - L group , MAT - H group and DEX group was lower than that in model group , but the difference was not statistically significant ( p < 0.05 ) . There was no significant difference among the three drug intervention groups ( p < 0.05 ) .

2 Weight changes : The average body weight increased steadily in the normal control group from the second day , and the average body weight in the remaining four groups showed a decrease trend . The body weight loss of the two MAT groups was significantly lower than that in the model group ( p 0.01 ) , which was also lower than that of the dexamethasone group ( p 0.01 ) . Compared with MAT - L group , the mean body weight change of MAT - H group was small , and the difference was statistically significant ( p 0.05 ) .

3 Neurologic score : The average nerve score of the normal control group was maintained at 0 . The mean neurological score in the model group was significantly higher than that of the three drug intervention groups ( p 0.05 ) on the 17th day after immunization . There was no significant difference between the three groups ( p < 0.05 ) .

4 histopathological findings : The mean inflammatory infiltration score and myelination score in the model group were significantly higher than those in the normal control group ( p 0.01 ) . The mean inflammatory infiltration score and myelination score of MAT - L group were significantly lower than that of model group ( p 0.01 ) . The mean inflammatory infiltration score and myelination score of MAT - H group were further reduced compared to MAT - L group ( p 0.05 ) . Compared with MAT - H group , the mean inflammatory infiltration score of DEX group was significantly lower ( p 0.01 ) , and there was no significant difference in myelination ( p < 0.05 ) .

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