苦参素对实验性自身免疫性脑脊髓炎大小鼠巨噬细胞免疫调节作用机制研究

发布时间：2018-05-12 08:19

本文选题：实验性自身免疫性脑脊髓炎 + 苦参素　；参考：《郑州大学》2017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)经典动物模型-实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)急性大鼠模型和慢性进行性小鼠的疾病症状和病理学影响,检测EAE大鼠体内少突胶质祖细胞(OPCs,NG2)增殖与成熟髓鞘细胞表面标志物(MBP)表达情况,巨噬细胞/小胶质细胞中炎性M1型CD68+i NOS+与抗炎性M2型CD68+Arg1+的共表达情况,以及营养因子NT3、IGF-1、LIF与JAK2/STAT3信号传导通路的变化情况;观察EAE小鼠脊髓中神经营养因子NT3的来源,及NT3+i NOS+和NT3+Arg1+共表达情况。探究苦参素对急性期EAE大鼠模型和慢性进行性EAE小鼠的巨噬细胞/小胶质细胞的免疫调节作用机制,旨在为MS患者寻找一种安全、经济、有效的治疗药物提供实验依据。方法:1 1.1建立急性EAE大鼠模型:无菌配置豚鼠全脊髓匀浆(guinea pig spinal cord homogenate,GPSCH),加入等量弗氏完全佐剂(complete freund adjuvant,CFA,含6mg/ml卡介苗)制成稳定油包水型抗原乳剂并免疫诱导6~8周龄雌性Wistar大鼠制备急性模型。1.2大鼠分组与干预:采用随机数字表法将30只免疫后的雌性Wistar大鼠随机分为EAE模型组(Vehicle)、MAT预防组(MAT-P)、MAT治疗组(MAT-T),每组10只。同时选取10只健康雌性Wistar大鼠作为正常组(Normal)。MAT-P组大鼠于免疫后第1天起每日1次给予6.7 ml/kg(250 mg/kg)MAT腹腔注射治疗,MAT-T组大鼠在发病初期(免疫后第11天)起每日1次给予6.7 ml/kg(250 mg/kg)MAT腹腔注射治疗,Normal组和Vehicle组大鼠于免疫后第1天起每日1次给予等量生理盐水替代治疗,四组大鼠均干预至第17天。2 2.1建立慢性进行性EAE小鼠模型:采用MOG35-55(10 mg)溶解于2 ml生理盐水中,与含有2.5 mg/ml结核分枝杆菌的完全弗氏佐剂等体积混合,使用玻璃注射器将混合液在冰上反复抽打至完全混合,制成油包水型乳白色乳剂,静置10min后无分层为合格。将C57BL/6小鼠使用2%的戊巴比妥麻醉后,在脊柱背侧中线两侧,分四点皮下注射免疫(0.2 ml/只)。在免疫第0天和第2天,每只小鼠分别腹腔注射含200 ng百日咳毒素的PBS溶液,建立EAE小鼠模型。2.2小鼠分组及干预措施:采用随机数字表法将60只8-10周用MOG35-55免疫后的C57BL/6小鼠分为两组:EAE模型组(Vehicle)和MAT干预组(MAT),每组30只。自EAE小鼠发病初期(免疫后第13天)开始给予MAT组小鼠每日1次腹腔注射6.7 ml/kg(200 mg/kg)苦参素注射液,Vehicle组小鼠给予等量生理盐水替代治疗,两组小鼠均干预至发病高峰期(免疫后第22天)。3收集标本:在免疫后第18天(大鼠)和23天(小鼠),麻醉动物,快速心尖采血取血清后进行心脏灌注,收集大小鼠脊髓和脑,部分用4%多聚甲醛固定后制成石蜡切片,其余-80℃冻存备用。4相关指标检测:苏木素-伊红(Hematoxylin Eosin,HE)染色与Luxol Fast Blue(LFB)染色分别观察CNS炎性细胞浸润、髓鞘脱失情况,逆转录聚合酶链反应法(RT-PCR法)分析NT3、IGF-1、LIF和JAK2含量,Western Blot法分析Arg-1、i NOS含量,免疫荧光单染学法分析NT3、MBP表达,免疫荧光双染分析NG2+PCNA+、CD68+i NOS+、CD68+Arg1+、NG2+NT3+、GFAP+NT3+、CD68+NT3+、NT3+i NOS+、NT3+Arg1+表达,酶联免疫吸附法(ELISA法)检测STAT3水平。主要研究结果分为以下三部分第一部分:苦参素对急性EAE大鼠和慢性进行性EAE小鼠临床症状与病理症状的影响。1发病率:Normal组大鼠未观察到发病情况,Vehicle组大鼠有9只发病,发病率为90%,MAT-T组大鼠有6只发病,发病率为60%,MAT-P组有5只发病,发病率为50%。Fisher确切概率分析结果示:Normal组大鼠发病率显著低于Vehicle组(p0.01);MAT-T组和MAT-P组大鼠发病率均明显低于Vehicle组,差异具有统计学意义(p0.05)。此外两个MAT组间不存在显著性差异(p0.05)。Vehicle组小鼠有26只发病,发病率86.67%,MAT组小鼠有22只发病,发病率73.33%,两组小鼠发病率间差异不具有统计学意义(p0.05)。2体重变化情况:Normal组大鼠自免疫后第1天起体重平稳上升,其余三组大鼠体重整体上均呈先上升后下降趋势。MAT-T组和MAT-P组大鼠体重降低幅度均显著低于Vehicle组大鼠(p0.01)。与MAT-T组大鼠相比,MAT-P组大鼠平均体重变化相对较小,且有统计学差异(p0.05)。EAE小鼠中Vehicle组与MAT组自免疫后平均体重均呈先稍微下降后上升再下降最后上升趋势,其中MAT组小鼠平均体重降低幅度显著低于Vehicle组小鼠(p0.01)。3神经功能学评分:Normal组大鼠平均神经评分一直为0。免疫后第18天,Vehicle组大鼠平均神经功能学评分显著高于MAT-T组与MAT-P组大鼠(p0.05)。MAT-T组大鼠神经功能评分显著高于MAT-P组(p0.05)。EAE小鼠免疫后第23天,Vehicle组小鼠平均神经功能学评分显著高于MAT组(p0.05)。4组织病理学情况:Normal组大鼠平均炎症浸润评分、髓鞘脱失评分和轴索损伤评分为0,Vehicle组大鼠平均炎症浸润评分、髓鞘脱失评分和轴索损伤评分较Noraml组明显升高,差异具有统计学意义(p0.01)。MAT-T组平均炎症浸润评分、髓鞘脱失评分和轴索损伤评分显著低于Vehicle组(p0.05)。与MAT-T组大鼠相比,MAT-P组大鼠平均炎症浸润、髓鞘脱失和轴索损伤评分均进一步降低(p0.05)。EAE小鼠中MAT组小鼠平均炎症浸润评分和髓鞘脱失评分均明显低于Vehicle组,差异具有显著性意义(p0.01)。第二部分:苦参素对急性EAE大鼠少突胶质祖细胞增殖、神经营养因子IGF-1、LIF、NT3和JAK2/STAT3通路的影响。1 EAE大鼠脑组织胼胝体中MBP和NG2+PCNA+的表达:与Normal组相比,Vehicle组MBP和NG2+PCNA+含量显著性降低(p0.01)。MAT-T组与MAT-P组MBP和NG2+PCNA+表达均较Vehicle组显著升高(p0.01),其中MAT-P组MBP和NG2+PCNA+的表达明显高于MAT-T组(p0.05)。2脊髓中IGF-1、LIF和NT3含量:Vehicle组较Normal组脊髓中IGF-1、LIF和NT3表达均显著降低(p0.01),而两个MAT组IGF-1、LIF和NT3表达水平与Vehicle组相比均呈显著上升(p0.01),其中MAT-P组上升幅度相对更加明显(p0.05)。3大鼠脊髓中JAK2含量与脑组织中STAT3含量:与Normal组相比,Vehicle组大鼠脊髓中JAK2和脑组织中STAT3表达均显著升高(p0.01)。两个MAT干预组大鼠JAK2与STAT3水平与Vehicle组相比均呈显著降低(p0.01),其中MAT-P组大鼠脊髓中JAK2和脑组织中STAT3水平降低幅度显著大于MAT-T组(p0.05)。第三部分:苦参素对慢性进行性EAE小鼠CNS中神经胶质细胞源性NT3和巨噬细胞/小胶质细胞的影响。1 EAE大鼠脑组织中CD68+i NOS+与CD68+Arg1+共表达水平:与Normal组相比,Vehicle组大鼠胼胝体中CD68+i NOS+共表达情况明显增加,且WB结果显示i NOS蛋白含量明显升高(p0.01)。两个MAT干预组中CD68+i NOS+共表达情况则明显降低,且WB结果显示i NOS蛋白水平均较Vehicle组显著性降低(p0.01),其中MAT-P组降低幅度更为明显(p0.01)。与Normal组相比,Vehicle组大鼠胼胝体中CD68+Arg1+共表达情况明显降低,且WB结果显示Arg1蛋白含量明显降低(p0.01)。两个MAT干预组中CD68+i NOS+共表达情况则明显升高,且WB结果显示Arg1蛋白水平均较Vehicle组显著性升高(p0.01),其中MAT-P组增加幅度较MAT-T组更为明显(p0.01)。2 EAE小鼠脊髓组织中NT3、i NOS和Arg1水平:与Vehicle组相比,MAT组小鼠i NOS含量显著降低(p0.001),而MAT组小鼠Arg1和NT3含量则显著升高(p0.05,p0.01)。3 EAE大鼠脑组织胼胝体中CD68+i NOS+和CD68+Arg1+共表达水平:与Normal组相比,Vehicle组大鼠CD68+i NOS+共表达水平明显升高(p0.01)。两个MAT干预组CD68+i NOS+共表达水平均较Vehicle组显著性降低(p0.01),其中MAT-P组降低幅度更为明显(p0.01)。Vehicle组较Normal组CD68+Arg1+共表达水平明显降低(p0.01)。MAT-T组和MAT-P组CD68+Arg1+共表达水平均较Vehicle组显著性增高(p0.01),且MAT-P组较MAT-T组CD68+Arg1+水平高,差异具有统计学意义(p0.01)。4 EAE小鼠脊髓中NT3+NG2+、NT3+GFAP+、NT3+CD68+共表达情况:与Vehicle组相比,MAT组小鼠脊髓中NT3+NG2+、NT3+CD68+、NT3+GFAP+共表达水平均显著升高(p0.05,p0.01,p0.001),且NT3+NG2+、NT3+CD68+、NT3+GFAP+共表达水平增加幅度依次上升。5 EAE小鼠脊髓中NT3+i NOS+和NT3+Arg1+共表达情况:与Vehicle组相比,MAT组脊髓中NT3+i NOS+表达明显降低(p0.01),而MAT组脊髓中NT3+Arg1+共表达则显著升高(p0.001)。结论MAT对急性EAE大鼠和慢性进行性EAE小鼠具有良好的防治作用,可能是通过下调JAK2/STAT3通路的表达,上调神经胶质细胞中神经营养因子NT3的分泌,促进巨噬细胞和小胶质细胞由促炎性M1型向抗炎性M2型的转化,提高神经元生存微环境,从对急性EAE大鼠和慢性进行性EAE小鼠起到良好的防治效果。
[Abstract]:Objective: To observe the effects of Matrine (MAT) on the classical animal model of multiple sclerosis (multiple sclerosis, MS) - experimental autoimmune encephalomyelitis (experimental autoimmune encephalomyelitis, EAE) acute rat model and chronic progressive mice, the pathological and pathological effects of the disease, and the detection of oligodendrocyte progenitor cells in EAE rats. (OPCs, NG2) the expression of the proliferation and mature myelin cell surface markers (MBP), the co expression of inflammatory M1 CD68+i NOS+ and anti inflammatory M2 CD68+Arg1+ in macrophage / microglia, and the alteration of the nutritional factors NT3, IGF-1, LIF and JAK2/STAT3 signal transduction pathways. Source, NT3+i NOS+ and NT3+Arg1+ co expression. To explore the immunomodulatory mechanism of Matrine on acute phase EAE rat model and chronic progressive EAE mouse macrophage / microglia, and to provide a basis for finding a safe, economical and effective therapeutic drug for MS patients. Methods: 11.1 an acute EAE rat model was established. Guinea pig spinal cord homogenate (GPSCH) was added to the whole spinal cord of guinea pigs. The stable oil coated water antigen emulsion was prepared by adding the equivalent Freund complete adjuvant (complete Freund adjuvant, CFA, 6mg/ml BCG) and immunized to induce the acute model of female rats with 6~8 weeks. 30 female Wistar rats were randomly divided into EAE model group (Vehicle), MAT prevention group (MAT-P), MAT treatment group (MAT-T) and 10 rats in each group. At the same time, 10 healthy female Wistar rats were selected as normal group (Normal).MAT-P group, and 6.7 ml/kg (250 mg/kg) were given 1 times a day after first days of immunization. At the initial stage (eleventh days after immunization), 6.7 ml/kg (250 mg/kg) MAT were given 1 times a day for intraperitoneal injection, and the rats in group Normal and Vehicle were given equal amount of saline replacement therapy for 1 times a day after first days of immunity. The four groups of rats were intervened to establish a chronic progressive EAE mouse model at seventeenth days, with MOG35-55 (10 mg) dissolved in 2. In ml normal saline, mixed with a complete Freund's adjuvant containing 2.5 mg/ml Mycobacterium tuberculosis, a glass syringe was used to beat the mixture into a complete mixture on the ice to make the oil water milk white emulsion. After the static 10min, no stratification was qualified. The C57BL/6 mice were anesthetized with 2% pentobarbital and in the dorsal midline of the spine. On both sides, immunization (0.2 ml/) was subcutaneously injected at four points. In the zeroth and second days of immunization, each mouse was intraperitoneally injected with PBS solution containing 200 ng pertussis toxin, and the.2.2 mice of the EAE mouse model were divided into groups and intervention measures: the 60 8-10 weeks C57BL/6 mice immunized with MOG35-55 were divided into two groups: EAE model group (Vehi). CLE) and MAT intervention group (MAT), 30 in each group. From the early onset of EAE mice (thirteenth days after immunization), the MAT group mice were given 6.7 ml/kg (200 mg/kg) Marine Injection every day. The Vehicle mice were given the equivalent saline replacement therapy. The two groups were all intervened to the peak period of the onset (twenty-second days after immunization). Eighteenth days after the epidemic (rats) and 23 days (mice), the anaesthetized animals, the rapid apex blood sampling to take the blood serum for heart perfusion, collecting the spinal cord and brain of the rats, partially fixed with 4% polyformaldehyde to make paraffin sections, and the remaining -80 degrees centigrade for.4 Related Indexes Detection: hematoxylin Yi Hong (Hematoxylin Eosin, HE) staining and Luxol Fast Blue (LFB) staining The infiltration of inflammatory cells and the loss of myelin sheath were observed, and the contents of NT3, IGF-1, LIF and JAK2 were analyzed by reverse transcription polymerase chain reaction (RT-PCR). Western Blot method was used to analyze Arg-1, I NOS content, immunofluorescence staining and immunofluorescence analysis. +i NOS+, NT3+Arg1+ expression, enzyme linked immunosorbent assay (ELISA method) detection of STAT3 level. The main results are divided into the following three parts: the first part of the study: the effect of Matrine on the clinical symptoms and pathological symptoms of acute EAE rats and chronic progressive EAE mice, the incidence of.1: the Normal group was not observed, and there were 9 rats in the Vehicle group. The incidence was 90% in group MAT-T, 6 in group MAT-T and 5 in group MAT-P. The incidence of the incidence was 50%.Fisher exact probability analysis. The incidence of Normal rats was significantly lower than that in Vehicle group (P0.01); the incidence of MAT-T and MAT-P rats was significantly lower than that in group Vehicle (P0.05). In addition, two MAT groups There was no significant difference (P0.05) between 26 mice in group.Vehicle and 86.67% of the incidence, 22 in MAT group and 73.33% in the incidence rate of the two groups. The difference between the two groups was not statistically significant (P0.05), the body weight of the Normal group increased steadily from the day of the immunization, and the body weight of the other three groups was all on the whole. The weight decrease of.MAT-T group and MAT-P group was significantly lower than that of group Vehicle (P0.01). Compared with the MAT-T group, the average weight of the MAT-P group was relatively small, and there was a statistically significant difference (P0.05) the average weight of Vehicle group and MAT group in.EAE mice was slightly decreased and then increased then. The average weight reduction of group MAT mice was significantly lower than that in group Vehicle (P0.01).3 neurologic score: the average nerve score of group Normal was 0. after eighteenth days, and the average neurologic score of group Vehicle was significantly higher than that in group MAT-T and MAT-P group rats (P0.05).MAT-T group. The score was significantly higher than that in group MAT-P (P0.05).EAE mice twenty-third days after immunization. The average neurologic score of group Vehicle mice was significantly higher than that in group MAT (P0.05).4 histopathology: the average inflammatory infiltration score, the myelin loss score and the axonal injury score in the Normal group were 0, the average inflammatory infiltration score in the group Vehicle, the myelin loss score and the score of the myelin sheath, and the score of the myelin loss and the loss of myelin. The score of axonal injury was significantly higher than that in the Noraml group. The difference was statistically significant (P0.01) the average inflammatory infiltration score in the group.MAT-T, the myelin loss score and the axonal injury score were significantly lower than that of the Vehicle group (P0.05). Compared with the MAT-T group, the average inflammatory infiltration in the group of MAT-P rats, the myelin loss and axonal injury scores were further reduced (P0.05).EAE The average inflammatory infiltration score and the myelin loss score in the MAT group were significantly lower than that in the Vehicle group, and the difference was significant (P0.01). The second part: the effect of Matrine on oligodendrocyte progenitor cell proliferation in acute EAE rats, the effects of neurotrophic factor IGF-1, LIF, NT3 and JAK2/STAT3 pathway on MBP and NG2+PC in the corpus callosum of.1 EAE rat brain tissue The expression of NA+: compared with group Normal, the content of MBP and NG2+PCNA+ in group Vehicle decreased significantly (P0.01), the expression of MBP and NG2+PCNA+ in.MAT-T group and MAT-P group was significantly higher than that in Vehicle group (P0.01). The expression of F and NT3 decreased significantly (P0.01), while the expression level of IGF-1, LIF and NT3 in the two MAT groups was significantly higher than that of the Vehicle group (P0.01), and the increasing amplitude of the MAT-P group was more obvious (P0.05) in the spinal cord of the.3 rats and the content of the brain tissue. The levels of JAK2 and STAT3 in the two MAT intervention groups were significantly lower than those in the Vehicle group (P0.01). The decrease of STAT3 level in the JAK2 and brain tissues in the spinal cord of the MAT-P group was significantly greater than that in the MAT-T group (P0.05). The third part: the neuroglial cells derived from the chronic progressive EAE mice and in the chronic progressive EAE mice. The effect of macrophage / microglia on the co expression of CD68+i NOS+ and CD68+Arg1+ in the brain tissue of.1 EAE rats: compared with the Normal group, the co expression of CD68+i NOS+ in the corpus callosum of the Vehicle group was significantly increased, and the WB results showed a significant increase in the I NOS protein content. The co expression of the CD68+i was obvious in the two intervention groups. The WB results showed that the level of I NOS protein was significantly lower than that of the Vehicle group (P0.01), and the decrease in MAT-P group was more obvious (P0.01). The co expression of CD68+Arg1+ in the corpus callosum in the Vehicle group was significantly lower than that in the Normal group, and the WB results showed that the Arg1 protein content was significantly reduced. The WB results showed that the level of Arg1 protein was significantly higher than that in the Vehicle group (P0.01), and the increase in the MAT-P group was more obvious than that in the MAT-T group (P0.01) in the spinal cord of.2 EAE mice. The level of co expression of CD68+i NOS+ and CD68+Arg1+ in the corpus callosum of.3 EAE rats was significantly increased (P0.05, P0.01). The level of CD68+i NOS+ co expression in the Vehicle group was significantly higher than that in the Normal group (P0.01). The level of co expression in the two intervention groups was significantly lower than that of the Normal group. The co expression level of CD68+Arg1+ in group Normal was significantly lower than that of group Normal (P0.01) in group.Vehicle (P0.01), and CD68+Arg1+ co expression level in group.MAT-T and MAT-P group was significantly higher than that in group Vehicle (P0.01), and MAT-P group was higher than that of MAT-T group. As compared with the Vehicle group, the co expression level of NT3+NG2+, NT3+CD68+ and NT3+GFAP+ in the spinal cord of group MAT mice increased significantly (P0.05, P0.01, p0.001), and the increase of NT3+NG2+, NT3+CD68+, NT3+GFAP+ co expression level in the spinal cord of the mice was increased in turn. The expression of NOS+ was significantly decreased (P0.01), while the co expression of NT3+Arg1+ in the spinal cord of MAT group was significantly increased (p0.001). Conclusion MAT has a good preventive effect on acute EAE rats and chronic progressive EAE mice. It may be through down regulation of the expression of JAK2/STAT3 pathway, up regulation of the secretion of neurotrophic factor NT3 in glial fine cells, and promoting macrophage and macrophage. The transformation of microglia from proinflammatory M1 type to anti inflammatory M2 type improves the survival microenvironment of neurons, and has a good effect on the prevention and treatment of acute EAE rats and chronic progressive EAE mice.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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