青藤碱衍生物4EDB的合成、鉴定以及对实验性自身免疫性心肌炎治疗作用的研究

发布时间：2018-04-13 23:35

本文选题：青藤碱衍生物4EDB + 青藤碱　；参考：《江苏大学》2017年硕士论文

【摘要】：目的:以青藤碱(sinomenine,SIN)为原料,合成一种新的青藤碱衍生物—4EDB,并观察青藤碱衍生物4EDB对实验性自身免疫性心肌炎(experimental autoimmune myocarditis,EAM)的治疗效果,同时对4EDB抗炎机制进行初步研究。方法:1.青藤碱衍生物4EDB的合成、鉴定以及抗炎活性检测:对青藤碱A环4号位置进行修饰,合成青藤碱衍生物4EDB,并通过氢谱和质谱进行鉴定,利用高效液相色谱法(hi gh performance liquid chromatography,HPLC)检测4EDB的纯度。建立脓毒血症小鼠模型,给予不同浓度青藤碱和4EDB进行灌胃治疗,观察小鼠的生存率,初步评价抗炎效果。2.4EDB对EAM的治疗作用:建立EAM模型小鼠,再次免疫以后用4EDB进行小鼠灌胃治疗并观察小鼠状态。21天后处死小鼠,取出心肌组织,进行HE染色与天狼腥红染色以及提取RNA。通过HE染色获取病理评分,天狼腥红染色评价纤维化,实时定量荧光PCR(quantitative real-time PCR,qRT-PCR)检测心肌组织中的炎症因子IL-lβ、IL-6、IL-17A和TGF-β表达水平。取小鼠眼球血,离心收集上清,利用酶联免疫吸附试验(enzymes linked immunosorbent assay,ELISA)检测小鼠血清中炎症因子IL-lβ、IL-6、IL-17A、IL-4和IFN-γ的表达水平。3.4EDB治疗EAM的机制探讨:以RAW264.7细胞为研究对象,利用流式细胞仪(flow cytometry,FCM)检测4EDB对巨噬细胞凋亡的影响;利用Transwell实验检测4EDB对巨噬细胞趋化的影响。结果:1.成功制备青藤碱衍生物4EDB,纯度达到99.59%;通过脓毒血症模型实验发现4EDB处理组小鼠的存活率高于青藤碱组,并且5 mg/kg的4EDB处理组抗炎效果较优;2.4EDB治疗EAM模型小鼠:通过心肌组织病理评分,我们发现5 mg/kg的4EDB能够缓解EAM的进程。ELISA和qRT-PCR实验结果显示4EDB可以明显减少EAM小鼠血清中炎症因子IL-17A、IL-lβ、IL-6的含量,而且天狼腥红染色结果显示4EDB能够缓解EAM小鼠的心肌纤维化程度;3.流式细胞仪检测和Transwell结果显示10 nmol/mL及其以下浓度的4EDB不仅能够诱导RAW264.7细胞的凋亡,也能够抑制RAW264.7细胞的迁移。结论:1.合成一种新的青藤碱衍生物4EDB,且纯度达到99.59%;与青藤碱相比,4EDB具有较好的抗炎效果;2.4EDB能够下调炎症因子IL-lβ、IL-6、IL-17A的水平,缓解心肌纤维化进展,从而缓解EAM小鼠心肌组织的损伤;3.4EDB对EAM的治疗作用可能是通过诱导心肌组织巨噬细胞凋亡以及抑制单核/巨噬细胞迁移发挥免疫抑制作用。
[Abstract]:Aim: to synthesize a new sinomenine derivative -4EDBs from sinomenine sinensis, and to observe the therapeutic effect of sinomenine derivative 4EDB on experimental autoimmune myocarditis, and to study the mechanism of 4EDB anti-inflammation.Method 1: 1.Synthesis, Identification and Anti-inflammatory activity Detection of Sinomenine Derivatives 4EDB: sinomenine Derivatives 4EDBs were synthesized by modifying the position of Sinomenine A Ring 4, and identified by hydrogen spectrum and mass spectrometry.High performance liquid chromatography (HPLC) was used to determine the purity of 4EDB.The sepsis mice model was established and the mice were treated with different concentrations of sinomenine and 4EDB by gavage. The survival rate of mice was observed, and the anti-inflammatory effect of 2.4EDB on EAM was preliminarily evaluated.After the second immunization, the mice were treated with 4EDB by gavage and the mice were killed after 21 days. The myocardial tissue was taken out, HE staining and Anabaena staining were performed, and the RNA was extracted.Pathological scores were obtained by HE staining, fibrosis was evaluated by Anabaena red staining, and the expression levels of IL-l 尾 -IL-6 IL-17A and TGF- 尾 in myocardial tissue were detected by real-time quantitative fluorescence PCR(quantitative real-time PCR qRT-PCR.Mouse eyeball blood was collected by centrifugation and supernatant was collected by centrifugation. The expression level of IL-l 尾 -IL-6, IL-17, IL-4 and IFN- 纬 in serum of mice was detected by enzyme-linked immunosorbent assay (Elisa) and enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay). The mechanism of EAM treated by 3.4EDB was discussed.The effect of 4EDB on macrophage apoptosis and the effect of 4EDB on macrophage chemotaxis were detected by flow cytometry (FCM) and Transwell assay respectively.The result is 1: 1.Sinomenine derivative 4EDBs were successfully prepared with purity of 99.59.The survival rate of mice treated with 4EDB was higher than that of sinomenine treated mice by sepsis model experiment.And the anti-inflammatory effect of 5 mg/kg 4EDB treatment group was better than that of 2.4EDB treatment of EAM model mice.We found that 5 mg/kg 4EDB could alleviate the progress of EAM. Elisa and qRT-PCR results showed that 4EDB could significantly reduce the content of IL-17AfIL-l 尾 -IL-6 in the serum of EAM mice, and the results of Tianlang Anabarene staining showed that 4EDB could alleviate the degree of myocardial fibrosis in EAM mice.The results of flow cytometry and Transwell showed that 10 nmol/mL or less 4EDB could not only induce the apoptosis of RAW264.7 cells, but also inhibit the migration of RAW264.7 cells.Conclusion 1.A new sinomenine derivative, 4EDBs, was synthesized and its purity was 99.590.Compared with sinomenine, 4EDB had a better anti-inflammatory effect. 2.4EDB could down-regulate the level of IL-l 尾 -IL-6IL-17A and alleviate the progression of myocardial fibrosis.The therapeutic effect of 3.4 EDB on EAM in EAM mice may be mediated by inducing myocardial macrophage apoptosis and inhibiting monocyte / macrophage migration.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R542.21

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